Growth hormone releasing peptides

ABSTRACT

Disclosed are peptide and peptidomimetic compounds generally according to formula (I), and pharmaceutically acceptable salts thereof, that are useful as GHRP analogs: R 1 -A 2 -A 3 -A 4 -A 5 -R 2  (I) or a pharmaceutically acceptable salt thereof, wherein: A 1  is Aib, Apc or Inp; A 2  is D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-Ser(Bzl), or D-Trp; A 3  is D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-Ser(Bzl), or D-Trp; A 4  is 2Fua, Om, 2Pal, 3Pal, 4Pal, Pff, Phe, Pim, Taz, 2Thi, 3Thi, Thr(Bzl); A 5  is Apc, Dab, Dap, Lys, Orn, or deleted; R 1  is hydrogen, (C1-6)alkyl, (C5-14)aryl, (C1-6)alkyl(C5-14)aryl, (C3-8)cycloakyl, or (C2-10)acyl; and R 2  is OH or NH 2 ; and pharmaceutical compositions and methods of use thereof.

This application is a United States national stage filing under 35U.S.C. §371 of international (PCT) application No. PCT/US2003/024834,filed Aug. 8, 2003 and designating the US, which claims priority to U.S.provisional application 60/402,263 filed Aug. 9, 2002.

BACKGROUND OF THE INVENTION

The pulsatile release of growth hormone from the pituitary somatotropsis regulated by two hypothalamic neumpeptides growth hormone-releasinghormone and somatostatin. Growth hormone-releasing hormone stimulatesrelease of growth hormone, whereas, somatostatin inhibits secretion ofgrowth hormone. (Frohman et al., Endocr. Rev. 1986, 7, 223-253, andStrobi et al., Pharmacol. Rev. 1994, 46, 1-34.)

Release of growth hormone from the pituitary somatotrops can also becontrolled by growth hormone-releasing peptides (GHRP's). A hexapeptide,His-D-Trp-Ala-Trp-D-Phe-Lys-amide (GHRP-6), was found to release growthhormone from somatotrops in a dose-dependent manner in several speciesincluding man. (Bowers et al., Endocrinology 1984, 114, 1537-1545.)Subsequent chemical studies on GHRP-6 led to the identification of otherpotent growth hormone secretagogues such as GHRP-1, GHRP-2 and hexarelin(Cheng et al., Endocrinology 1989, 124, 2791-2798, Bowers, C. Y. NovelGH-Releasing Peptides. In: Molecular and Clinical Advances in PituitaryDisorder Ed: Melmed, S.; Endocrine Research and Education, Inc., LosAngeles, Calif., USA 1993, 153-157, and Deghenghi et al., Life Sci.1994, 54, 1321-1328):

-   -   GHRP-1 Ala-His-D-(2′)-Nal-Ala-Trp-D-Phe-Lys-NH₂;    -   GHRP-2 D-Ala-D-(2′)-Nal-Ala-Trp-D-Nal-Lys-NH₂;    -   Hexarelin His-D-2-MeTrp-Ala-Trp-D-Phe-Lys-NH₂;

GHRP-1, GHRP-2, GHRP-6, and hexarelin are synthetic growth hormonesecretagogues (GHS's). GHS's stimulate secretion of growth hormone by amechanism different from that of growth hormone-releasing hormone.(Bowers et al., Endocrinology 1984, 114, 1537-1545, Cheng et al.,Endocrinology 1989, 124, 2791-2798, Bowers, C. Y. Novel GH-ReleasingPeptides. In: Molecular and Clinical Advances in Pituitary Disorders.Ed: Melmed, S.; Endocrine Research and Education, Inc., Los Angeles,Calif., USA 1993, 153-157, and Deghenghi et al., Life Sci. 1994, 54,1321-1328.)

The low oral bioavailability (<1%) of the peptidyl growth hormonesecretagogues stimulated search for non-peptide compounds mimickingaction of GHRP-6 in the pituitary. Several benzolactams and spiroindaneshave been reported to stimulate growth hormone release in various animalspecies and in man. (Smith et al. Science 1993, 260, 1640-1643, Patchettet al., Proc. Natl. Acad. Sci. USA. 1995, 92, 7001-7005, and Chen etal., Bioorg. Mod. Chem. Lett. 1996, 6, 2163-2169.) A specific example ofa small spiroindane is MK-0677 (Patchett et al. Proc. Natl. Acad. Sci.USA. 1995, 92, 7001-7005):

The actions of the above-mentioned GHS's (both peptide and non-peptide)appear to be mediated by a specific growth hormone secretagogue receptor(GHS receptor). (Howard et al., Science 1996, 273,974-977, and Pong etal, Molecular Endocrinology 1996, 10, 57-61.) This receptor is presentin the pituitary and hypothalamus of various mammalian species (GHSR1a)and is distinct from the growth hormone-releasing hormone (GHRH)receptor. The GHS receptor was also detected in the other areas of thecentral nervous system and In peripheral tissues, for instance adrenaland thyroid glands, heart, lung, kidney and skeletal muscles. (Chen etal, Bioorg. Med. Chem Lett. 1996, 6, 2163-2169, Howard et al, Science1996, 273, 974-977, Pong et al, Molecular Endocrinology 1996, 10, 57-61,Guan et al, Mol. Brain Res. 1997, 48, 23-29, and McKee et al., Genomics1997, 46, 426-434.) A truncated version of GHSR1a has been reported.(Howard et al., Science 1996, 273, 974-977.)

The GHS receptor is a G-protein coupled-receptor. Effects of GHSreceptor activation include depolarization and inhibition of potassiumchannels, an increase in intercellular concentrations of inositoltriphosphate (IP3), and a transient increase in the concentrations ofintracellular calcium. (Pong et al., Molecular Endocrinology 1996, 10,57-61, Guan et al., Mol. Brain Res. 1997, 48, 23-29, and McKee et al,Genomics 1997, 46, 426-434.)

Ghrelin is a naturally occurring peptide which is believed to be anendogenous ligand for the GHS receptor. (Kojima et al., Nature 1999,402, 656-660.) The native structures of ghrelins from several mammalianand non-mammalian species of animals are known. (Kalya et al., J. Biol.Chem. 2001, 276, 40441-40448; International Patent ApplicationPCT/JP00/04907 (WO 01/07475)) A core region present in ghrelin was foundto provide for activity at the GHS receptor. The core region comprisesthe four N-terminal amino acids, where the serine at position 3 isnormally modified with n-octanoic. However, in addition to acylation byn-octanoic acid native ghrelin also has been observed to be acylatedwith n-decanoic acid. (Kalya et al., J. Biol. Chem. 2001, 276,40441-40448.) Ghrelin analogs have a variety of different therapeuticuses as well as uses as research tools.

SUMMARY OF THE INVENTION

The present invention features peptidyl analogs active at the GHSreceptor. The analogs of the invention can bind to the GHS receptor and,preferably, bring about signal transduction.

Thus, in a first aspect the present invention features a compoundaccording to formula (I):R¹-A¹-A²-A³-A⁴-A⁵-R²  (I)or a pharmaceutically acceptable salt thereof, wherein:

-   -   A¹ is Aib, Apc or Inp;    -   A² is D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-Ser(Bzl), or        D-Trp;    -   A³ is D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-Ser(Bzl), or        D-Trp;    -   A⁴ is 2Fua, Orn, 2Pal, 3Pal, 4Pal, Pff, Phe, Pim, Taz, 2Thi,        3Thi, Thr(Bzl);    -   A⁵ is Apc, Dab, Dap, Lys, Orn, or deleted;    -   R¹ is hydrogen, (C₁₋₆)alkyl, (C₅₋₁₄)aryl, (C₁₋₆)alky,        (C₅₋₁₄)aryl, (C₃₋₈)cycloalkyl, or (C₂₋₁₀)acyl; and    -   R² is OH or NH₂;        provided that:    -   when A⁶ is Dab, Dap, Lys, or Orn, then:        -   A² is D-Bip, D-Bpa, D-Dip or D-Bal; or        -   A³ is D-Bip, D-Bpa, D-Dip or D-Bal; or        -   A⁴ is 2Thi, 3Thi, Taz, 2Fua, 2Pal, 3Pal, 4Pal, Orn,            Thr(Bzl), or Pff;    -   when A⁵ is deleted, then:        -   A³ is D-Bip, D-Bpa, or D-Dip; or        -   A⁴ is 2Fua, Pff, Taz, or Thr(Bzl); or        -   A¹ is Apc and            -   A² is D-Bip, D-Bpa, D-Dip or D-Bal; or            -   A³ is D-Bip, D-Bpa, D-Dip or D-Bal; or            -   A⁴ is 2Thi, 3Thi, Orn, 2Pal, 3Pal, or 4Pal.

A preferred compound of formula (I), termed a Group 1 compound, is acompound according to formula (I) wherein:

-   -   A¹ is Aib, Apc or Inp    -   A² is D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-Ser(Bzl), or        D-Trp;    -   A³ is D-Bal, D-Bpa, D-Dip, D-1Nal, D-2Nal, or D-Trp;    -   A⁴ is Orn, 3Pal, 4Pal, Pff, Phe, Pim, Taz, 2Thi, or Thr(Bzl);        and    -   A⁵ is Apc, Lys, or deleted;        or a pharmaceutically acceptable salt thereof.

A preferred Group 1 compound, termed a Group 1A compound, is a compoundaccording to the formula:

-   -   A¹ is Apc or Inp    -   A² is D-Bal, D-Bip, D-1Nal, or D-2Nal;    -   A³ is D-Bal, D-1Nal, D-2Nal, or D-Trp;    -   A⁴ is 3Pal, 4Pal, Pff, Phe, Pim, Taz, 2Thi, or Thr(Bzl); and or        a pharmaceutically acceptable salt thereof.

Another preferred compound of formula (I), termed a Group 2 compound, isa compound according to the formula:

-   -   H-Inp-D-1Nal-D-Trp-3Pal-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-4Pal-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Om-Lys-NH₂;    -   H-Inp-D-Bip-Trp-Phe-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Thr(Bzl)-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Pff-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Taz-Lys-NH₂;    -   H-Inp-D-Dip-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-Bpa-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-2Nal-D-Bpa-Phe-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-3Pal-NH₂;    -   H-Inp-D-2Nal-D-Trp-4Pal-NH₂;    -   H-Inp-D-1Nal-D-Trp-3Pal-NH₂;    -   H-Inp-D-Bip-D-Trp-Phe-NH₂;    -   H-Inp-D-2Nal-D-Trp-Thr(Bzl)-NH₂;    -   H-Inp-D-2Nal-D-Trp-Pff-NH₂;    -   H-Inp-D-2Nal-D-Trp-2Thi-NH₂;    -   H-Inp-D-2Nal-D-Trp-Taz-NH₂;    -   H-Inp-D-Dip-D-Trp-Phe-NH₂;    -   H-Inp-D-2Nal-D-Dip-Phe-NH₂;    -   H-Inp-D-Bal-D-Trp-Phe-NH₂;    -   H-Inp-D-2Nal-D-Bal-Phe-NH₂;    -   H-Inp-D-2Nal-D-Trp-3Pal-Lys-NH₂;    -   H-Inp-D-Trp-D-2Nal(Ψ)-Pim;    -   H-Inp-D-Bal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-1Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Phe-Apc-NH₂;    -   H-Inp-D-1Nal-D-Trp-Phe-Apc-NH₂;    -   H-Inp-D-Bal-D-Trp-Phe-Apc-NH₂;    -   H-Apc-D-2Nal-D-Trp-Phe-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-1Nal-D-Trp-2Thi-NH₂;    -   H-Apc-D-1Nal-D-Trp-Phe-NH₂;    -   H-Inp-D-2Nal-D-Trp(Ψ)-Pim;    -   H-Inp-D-1Nal-D-Trp(Ψ)-Pim;    -   H-Inp-D-Bal-D-Trp(Ψ)-Pim;    -   H-Aib-D-Ser(Bzl)-D-Trp(Ψ)-Pim;    -   H-Inp-D-1Nal-D-Trp-Taz-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-2Thi-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-Phe-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-Phe-Apc-NH₂;    -   H-Apc-D-Bal-D-Trp-Phe-Apc-NH₂;    -   H-Apc-D-1Nal-D-1Nal-Phe-Apc-NH₂;    -   H-Apc-D-1Nal-D-2Nal-Phe-Apc-NH₂;    -   H-Apc-D-1Nal-D-1Nal-Phe-Lys-NH₂;    -   H-Apc-D-Bal-D-1Nal-Phe-Apc-NH₂;    -   H-Apc-D-Bal-D-2Nal-Phe-Apc-NH₂;    -   H-Apc-D-Bal-D-1Nal-Phe-Lys-NH₂;    -   H-Apc-D-Bal-D-2Nal-Phe-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Thi-NH₂;    -   H-Apc-D-Bal-D-Trp-Phe-NH₂;    -   H-Apc-D-1Nal-D-Trp-Taz-NH₂;    -   H-Apc-D-Bal-D-Trp-2Thi-NH₂;    -   H-Apc-D-Bal-D-Trp-Taz-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Thi-NH₂;    -   H-Apc-D-2Nal-D-Trp-Taz-NH₂;    -   H-Inp-D-1Nal-D-Trp-Taz-Apc-NH₂;    -   H-Inp-D-Bal-D-Trp-Taz-Apc-NH₂;    -   H-Apc-D-1Nal-D-Trp-Taz-Apc-NH₂;    -   H-Apc-D-Bal-D-Trp-Taz-Apc-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Fua-Apc-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Fua-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Fua-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Pal-NH₂;    -   H-Apc-D-1Nal-D-Trp-3Pal-NH₂;    -   H-Apc-D-1Nal-D-Trp-3Thi-Apc-NH₂;    -   H-Apc-D-1Nal-D-Trp-3Thi-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-3Thi-NH₂;    -   H-Apc-D-1Nal-D-Trp-4Pal-NH₂;    -   H-Apc-D-1Nal-D-Trp-Pff-Apc-NH₂;    -   H-Apc-D-1Nal-D-Trp-Pff-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-Pff-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Fua-Apc-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Fua-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Fua-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Pal-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Thi-Apc-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Apc-D-2Nal-D-Trp-3Pal-NH₂;    -   H-Apc-D-2Nal-D-Trp-3Thi-Apc-NH₂;    -   H-Apc-D-2Nal-D-Trp-3Thi-Lys-NH₂;    -   H-Apc-D-2Nal-D-Trp-3Thi-NH₂;    -   H-Apc-D-2Nal-D-Trp-4Pal-NH₂;    -   H-Apc-D-2Nal-D-Trp-Pff-Apc-NH₂;    -   H-Apc-D-2Nal-D-Trp-Pff-Lys-NH₂;    -   H-Apc-D-2Nal-D-Trp-Pff-NH₂;    -   H-Apc-D-2Nal-D-Trp-Taz-Apc-NH₂;    -   H-Apc-D-2Nal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-Bal-D-Bal-2Fua-Apc-NH₂;    -   H-Apc-D-Bal-D-Bal-2Fua-Lys-NH₂;    -   H-Apc-D-Bal-D-Bal-2Fua-NH₂;    -   H-Apc-D-Bal-D-Bal-2Pal-NH₂;    -   H-Apc-D-Bal-D-Bal-2Thi-Apc-NH₂;    -   H-Apc-D-Bal-D-Bal-2Thi-Lys-NH₂;    -   H-Apc-D-Bal-D-Bal-2Thi-NH₂;    -   H-Apc-D-Bal-D-Bal-3Pal-NH₂;    -   H-Apc-D-Bal-D-Bal-3Thi-Apc-NH₂;    -   H-Apc-D-Bal-D-Bal-3Thi-Lys-NH₂;    -   H-Apc-D-Bal-3Thi-NH₂;    -   H-Apc-D-Bal-D-Bal-4Pal-NH₂;    -   H-Apc-D-Bal-D-Bal-Pff-Apc-NH₂;    -   H-Apc-D-Bal-D-Bal-Lys-NH₂;    -   H-Apc-D-Bal-D-Bal-Pff-NH₂;    -   H-Apc-D-Bal-D-Bal-Phe-Apc-NH₂;    -   H-Apc-D-Bal-D-Bal-Phe-Lys-NH₂;    -   H-Apc-D-Bal-D-Bal-Phe-NH₂;    -   H-Apc-D-Bal-D-Bal-Taz-Apc-NH₂;    -   H-Apc-D-Bal-D-Bal-Taz-Lys-NH₂;    -   H-Apc-D-Bal-D-Bal-Taz-NH₂;    -   H-Apc-D-Bal-D-Trp-2Fua-Apc-NH₂;    -   H-Apc-D-Bal-D-Trp-T2Fua-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-2Fua-NH₂;    -   H-Apc-D-Bal-D-T2Pal-NH₂;    -   H-Apc-D-Bal-D-Trp-3Pal-NH₂;    -   H-Apc-D-Bal-D-Trp-3Thi-NH₂;    -   H-Apc-D-Bal-D-Trp-3Thi-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-3Thi-NH₂;    -   H-Apc-D-Bal-D-Trp-4Pal-NH₂;    -   H-Apc-D-Bal-D-Trp-Pff-Apc-NH₂;    -   H-Apc-D-Bal-D-Trp-Pff-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-Pff-NH₂;    -   H-Inp-D-1Nal-D-Bal-2Fua-Lys-NH₂;    -   H-Inp-D-1Nal-D-Bal-2Fua-NH₂;    -   H-Inp-D-1Nal-D-Bal-2Thi-Lys-NH₂;    -   H-Inp-D-1Nal-D-Bal-3Thi-Lys-NH₂;    -   H-Inp-D-1Nal-D-Bal-Pff-Lys-NH₂;    -   H-Inp-D-1Nal-D-Bal-Pff-NH₂;    -   H-Inp-1Nal-D-Bal-Phe-Lys-NH₂;    -   H-Inp-1Nal-D-Bal-Taz-Lys-NH₂;    -   H-Inp-D-1Nal-D-Bal-Taz-NH₂;    -   H-Inp-D-1Nal-D-Trp-2Fua-Apc-NH₂;    -   H-Inp-D-1Nal-D-Trp-2Fua-Lys-NH₂;    -   H-Inp-D-Nal-D-Trp-2Fua-NH₂;    -   H-Inp-D-1Nal-D-Trp-3Thi-Apc-NH₂;    -   H-Inp-D-1Nal-D-Trp-3Thi-Lys-NH₂;    -   H-Inp-D-1Nal-D-Trp-Pff-Apc-NH₂;    -   H-Inp-D-1Nal-D-Trp-Pff-Lys-NH₂;    -   H-Inp-D-1Nal-D-Trp-Pff-NH₂;    -   H-Inp-D-1Nal-D-Trp-Taz-NH₂;    -   H-Inp-D-2Nal-D-Trp-2Fua-Apc-NH₂;    -   H-Inp-D-2Nal-D-Trp-2Fua-NH₂;    -   H-Inp-D-2Nal-D-Trp-2Thi-Apc-NH₂;    -   H-Inp-D-2Nal-D-Trp-3Thi-Apc-NH₂;    -   H-Inp-D-2Nal-D-Trp-3Thi-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-3Thi-NH₂;    -   H-Inp-D-2Nal-D-Trp-Pff-Apc-NH₂;    -   H-Inp-D-2Nal-D-Trp-Pff-NH₂;    -   H-Inp-D-2Nal-D-Trp-Taz-Apc-NH₂;    -   H-Inp-2Nal-D-Trp-Taz-NH₂;    -   H-Inp-D-Bal-D-Bal-2Fua-Lys-NH₂;    -   H-Inp-D-Bal-D-Bal-2Fua-NH₂;    -   H-Inp-D-Bal-D-Bal-2Thi-Lys-NH₂;    -   H-Inp-D-Bal-D-Bal-3Thi-Lys-NH₂;    -   H-Inp-D-Bal-D-Bal-Pff-Lys-NH₂;    -   H-Inp-D-Bal-D-Bal-Pff-NH₂;    -   H-Inp-D-Bal-D-Bal-Phe-Lys-NH₂;    -   H-Inp-D-Bal-D-Bal-Taz-Lys-NH₂:    -   H-Inp-D-Bal-D-Bal-Taz-NH₂;    -   H-Inp-D-Bal-D-Trp-2Fua-Apc-NH₂;    -   H-Inp-D-Bal-D-Trp-2Fua-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-2Fua-NH₂;    -   H-Inp-D-Bal-D-Trp-3Thi-Apc-NH₂;    -   H-Inp-D-Bal-D-Trp-3Thi-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-Pff-Apc-NH₂;    -   H-Inp-D-Bal-D-Trp-Pff-NH₂;    -   H-Inp-D-Bal-D-Trp-Pff-NH₂;    -   H-Inp-D-Bal-D-Trp-Taz-NH₂;    -   H-Inp-D-Bip-D-Bal-2Fua-Lys-NH₂;    -   H-Inp-D-Bip-D-Bal-2Fua-NH₂;    -   H-Inp-D-Bip-D-Bal-2Thi-Lys-NH₂;    -   H-Inp-D-Bip-D-Bal-3Thi-Lys-NH₂;    -   H-Inp-Bip-D-Bal-Pff-Lys-NH₂;    -   H-Inp-D-Bip-D-Bal-Pff-NH₂; or    -   H-Inp-D-Bip-D-Bal-Taz-Lys-NH₂;    -   H-Inp-Bip-D-Bal-Taz-NH₂;    -   H-Inp-D-Bip-D-Trp-2Fua-Lys-NH₂;    -   H-Inp-D-Bip-D-Trp-2Fua-NH₂;    -   H-Inp-D-Bip-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-Bip-D-Trp-3Thi-Lys-NH₂;    -   H-Inp-D-Bip-D-Trp-Pff-Lys-NH₂;    -   H-Inp-D-Bip-D-Trp-Pff-NH₂;    -   H-Inp-D-Bip-D-Trp-Taz-Lys-NH₂; or    -   H-Inp-D-Bip-D-Trp-Taz-NH₂;        or a pharmaceutically acceptable salt thereof.

A preferred Group 2 compound, termed a Group 2A compound, is a compoundaccording to the formula:

-   -   H-Inp-D-1Nal-D-Trp-3Pal-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-4Pal-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Om-Lys-NH₂;    -   H-Inp-D-Bip-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Thr(Bzl)-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Pff-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Taz-Lys-NH₂;    -   H-Inp-D-Dip-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-Bpa-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-2Nal-D-Bpa-Phe-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Thr-(Bzl)-NH₂;    -   H-Inp-D-2Nal-D-Trp-Pff-NH₂;    -   H-Inp-D-2Nal-D-Trp-Taz-NH₂;    -   H-Inp-D-2Nal-Dip-Phe-NH₂;    -   H-Inp-D-2Nal-D-Trp-3Pal-Lys-NH₂;    -   H-Inp-D-Trp-D-2Nal(Ψ)-Pim;    -   H-Inp-D-Bal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-1Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Phe-Apc-NH₂;    -   H-Inp-D-1Nal-D-Trp-Phe-Apc-NH₂;    -   H-Inp-D-Bal-D-Trp-Phe-Apc-NH₂;    -   H-Apc-D-2Nal-D-Trp-Phe-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp(Ψ)-Pim;    -   H-Inp-D-1Nal-D-Trp(Ψ)-Pim;    -   H-Inp-D-Bal-D-Trp(Ψ)-Pim;    -   H-Aib-D-Ser(Bzl)-D-Trp(Ψ)-Pim;    -   H-Inp-D-1Nal-D-Trp-Taz-Lys-NH₂;    -   H-Inp-D-Bal-Trp-Taz-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-Trp-2Thi-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-Phe-Lys-NH₂;    -   H-Apc-D-1Nal-Trp-Phe-Apc-NH₂;    -   H-Apc-D-Bal-D-Trp-Phe-Apc-NH₂;    -   H-Apc-D-1Nal-D-1Nal-Phe-Apc-NH₂;    -   H-Apc-D-1Nal-D-2Nal-Phe-Apc-NH₂;    -   H-Apc-D-1Nal-D-1Nal-Phe-Lys-NH₂;    -   H-Apc-D-Bal-D-1Nal-Phe-Apc-NH₂;    -   H-Apc-D-Bal-D-2Nal-Phe-Apc-NH₂;    -   H-Apc-D-Bal-D-1Nal-Phe-Lys-NH₂;    -   H-Apc-D-Bal-D-2Nal-Phe-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Thi-NH₂;    -   H-Apc-D-Bal-D-Trp-Phe-NH₂;    -   H-Apc-D-1Nal-D-Trp-Taz-NH₂;    -   H-Apc-D-Bal-D-Trp-2Thi-NH₂;    -   H-Apc-D-Bal-D-Trp-Taz-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Thi-NH₂;    -   H-Apc-D-2Nal-D-Trp-Taz-NH₂;    -   H-Inp-D-1Nal-D-Trp-Taz-Apc-NH₂;    -   H-Inp-D-Bal-D-Trp-Taz-Apc-NH₂;    -   H-Apc-D-1Nal-D-Trp-Taz-Apc-NH₂;    -   H-Apc-D-Bal-D-Trp-Taz-Apc-NH₂;    -   H-Inp-D-2Nal-D-Trp-3Thi-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-3Thi-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-2Fua-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-Pff-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-3Thi-Apc-NH₂;    -   H-Inp-D-Bal-D-Trp-2Fua-Apc-NH₂;    -   H-Inp-D-Bal-D-Trp-Pff-Apc-NH₂;    -   H-Apc-Bal-D-Trp-3Th-Lys-NH₂:    -   H-Apc-D-Bal-D-Trp-2Fua-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-Pff-Lys-NH₂;    -   H-Inp-D-Bal-D-Bal-Phe-Lys-NH₂;    -   H-Inp-D-Bal-D-Bal-2Thi-Lys-NH₂;    -   H-Inp-D-Bal-D-Bal-3Thi-Lys-NH₂;    -   H-Inp-D-Bal-D-Bal-Taz-Lys-NH₂;    -   H-Inp-D-Bal-D-Bal-2Fua-Lys-NH₂;    -   H-Inp-D-Bal-D-Bal-Pff-Lys-NH₂;    -   H-Apc-D-Bal-D-Bal-Phe-Lys-NH₂;    -   H-Apc-D-Bal-D-Bal-2Thi-Lys-NH₂;    -   H-Apc-D-Bal-D-Bal-3Thi-Lys-NH₂;    -   H-Apc-D-Bal-D-Bal-Taz-Lys-NH₂;    -   H-Apc-D-Bal-D-Bal-2Fua-Lys-NH₂;    -   H-Apc-D-Bal-D-Bal-Pff-Lys-NH₂;    -   H-Inp-D-1Nal-D-Trp-3Thi-Lys-NH₂;    -   H-Inp-D-1Nal-D-Trp-2Fua-Lys-NH₂;    -   H-Inp-D-1Nal-D-Trp-Pff-Lys-NH₂;    -   H-Inp-D-1Nal-D-Bal-Phe-Lys-NH₂;    -   H-Inp-D-1Nal-D-Bal-2Thi-Lys-NH₂;    -   H-Inp-D-1Nal-D-Bal-3Thi-Lys-NH₂;    -   H-Inp-D-1Nal-D-Bal-Taz-Lys-NH₂;    -   H-Inp-D-1Nal-D-Bal-2Fua-Lys-NH₂;    -   H-Inp-D-1Nal-D-Bal-Pff-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-2Thi-Apc-NH₂;    -   H-Inp-D-2Nal-D-Trp-3Thi-Apc-NH₂;    -   H-Inp-D-2-Nal-D-Trp-Taz-Apc-NH₂;    -   H-Inp-D-2-Nal-D-Trp-2Fua-Apc-NH₂;    -   H-Inp-D-2-Nal-D-Trp-Pff-Apc-NH₂;    -   H-Inp-D-1Nal-D-Trp-3Thi-Apc-NH₂;    -   H-Inp-D-1Nal-D-Trp-2Fua-Apc-NH₂;    -   H-Inp-D-1Nal-D-Trp-Pff-Apc-NH₂;    -   H-Apc-D-1Nal-D-Trp-3Thi-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Fua-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-Pff-Lys-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Apc-D-2Nal-D-Trp-3Thi-Lys-NH₂;    -   H-Apc-D-2Nal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Fua-Lys-NH₂;    -   H-Apc-D-2Nal-D-Trp-Pff-Lys-NH₂;    -   H-Inp-D-Bip-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-Bip-D-Trp-3Thi-Lys-NH₂;    -   H-Inp-D-Bip-D-Trp-Taz-Lys-NH₂;    -   H-Inp-D-Bip-D-Trp-2Fua-Lys-NH₂;    -   H-Inp-D-Bip-D-Trp-Pff-Lys-NH₂;    -   H-Inp-Bip-D-Bal-2Thi-Lys-NH₂;    -   H-Inp-D-Bip-D-Bal-3Thi-Lys-NH₂;    -   H-Inp-D-Bip-D-Bal-Taz-Lys-NH₂;    -   H-Inp-Bip-Bal-2Fua-Lys-NH₂;    -   H-Inp-D-Bip-D-Bal-Pff-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-3Thi-Apc-NH₂;    -   H-Apc-D-Bal-D-Trp-2Fua-Apc-NH₂;    -   H-Apc-D-Bal-D-Trp-Pff-Apc-NH₂;    -   H-Apc-D-Bal-D-Bal-Phe-Apc-NH₂;    -   H-Apc-D-Bal-D-Bal-2Thi-Apc-NH₂;    -   H-Apc-D-Bal-D-Bal-3Thi-Apc-NH₂;    -   H-Apc-D-Bal-D-Bal-Taz-Apc-NH₂;    -   H-Apc-D-Bal-D-Bal-2Fua-Apc-NH₂;    -   H-Apc-D-Bal-D-Bal-Pff-Apc-NH₂;    -   H-Apc-D-1Nal-D-Trp-3-Thi-Apc-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Fua-Apc-NH₂;    -   H-Apc-D-1Nal-D-Trp-Pff-Apc-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Thi-Apc-NH₂;    -   H-Apc-D-2Nal-D-Trp-3Thi-Apc-NH₂;    -   H-Apc-D-2Nal-D-Trp-Taz-Apc-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Fua-Apc-NH₂;    -   H-Apc-D-2Nal-D-Trp-Pff-Apc-NH₂;    -   H-Inp-D-Bal-D-Trp-Taz-NH₂;    -   H-Inp-D-Bal-D-Trp-2Fua-NH₂;    -   H-Inp-D-Bal-D-Trp-Pff-NH₂;    -   H-Apc-D-Bal-D-Trp-3Thi-NH₂;    -   H-Apc-D-Bal-Trp-2Fua-NH₂;    -   H-Apc-D-Bal-D-Trp-Pff-NH₂;    -   H-Apc-D-Bal-D-Trp-4Pal-NH₂;    -   H-Apc-D-Bal-D-Trp-3Pal-NH₂;    -   H-Apc-D-Bal-D-Trp-2Pal-NH₂;    -   H-Inp-D-Bal-D-Bal-Taz-NH₂;    -   H-Inp-D-Bal-D-Bal-2Fua-NH₂;    -   H-Inp-D-Bal-D-Bal-Pff-NH₂;    -   H-Apc-D-Bal-D-Bal-Phe-NH₂;    -   H-Apc-D-Bal-D-Bal-2Thi-NH₂;    -   H-Apc-D-Bal-D-Bal-3Thi-NH₂;    -   H-Apc-D-Bal-D-Bal-Taz-NH₂;    -   H-Apc-D-Bal-D-Bal-2Fua-NH₂;    -   H-Apc-D-Bal-D-Bal-Pff-NH₂;    -   H-Apc-D-Bal-D-Bal-Phe-NH₂;    -   H-Apc-D-Bal-D-Bal-3Pal-NH₂;    -   H-Apc-D-Bal-D-Bal-2Pal-NH₂;    -   H-Inp-D-1Nal-D-Trp-Taz-NH₂;    -   H-Inp-D-1Nal-D-Trp-2Fua-NH₂;    -   H-Inp-D-1Nal-D-Trp-Pff-NH₂;    -   H-Inp-D-1Nal-D-Trp-Taz-NH₂;    -   H-Inp-D-1Nal-D-Bal-2Fua-NH₂;    -   H-Inp-D-1Nal-D-Bal-Pff-NH₂;    -   H-Inp-D-2Nal-D-Trp-Taz-NH₂;    -   H-Inp-D-2Nal-D-Trp-2Fua-NH₂;    -   H-Inp-D-2Nal-D-Trp-Pff-NH₂;    -   H-Apc-D-1Nal-D-Trp-3Thi-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Fua-NH₂;    -   H-Apc-D-1Nal-D-Trp-Pff-NH₂;    -   H-Apc-D-1Nal-D-Trp-4Pal-NH₂;    -   H-Apc-D-1Nal-D-Trp-3Pal-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Pal-NH₂;    -   H-Apc-D-2Nal-D-Trp-3Thi-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Fua-NH₂;    -   H-Apc-D-2Nal-D-Trp-Pff-NH₂;    -   H-Apc-D-2Nal-D-Trp-4Pal-NH₂;    -   H-Apc-D-2Nal-D-Trp-3Pal-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Pal-NH₂;    -   H-Inp-D-Bip-D-Trp-Taz-NH₂;    -   H-Inp-D-Bip-D-Trp-2Fua-NH₂;    -   H-Inp-D-Bip-D-Trp-Pff-NH₂;    -   H-Inp-D-Bip-D-Bal-Taz-NH₂;    -   H-Inp-D-Bip-D-Bal-2Fua-NH₂; or    -   H-Inp-D-Bip-D-Bal-Pff-NH₂;        or a pharmaceutically acceptable salt thereof.

A preferred Group 2A compound, termed a Group 2B compound, is a compoundaccording to the formula:

-   -   H-Inp-D-1Nal-D-Trp-3Pal-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-4Pal-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Om-Lys-NH₂;    -   H-Inp-D-Bip-D-Trp-Phe-Lys-H₂;    -   H-Inp-D-2Nal-D-Trp-Thr(Bzl)-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Pff-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Taz-Lys-NH₂;    -   H-Inp-D-Dip-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-Bpa-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-2Nal-D-Bpa-Phe-Lys-NH₂;    -   H-Inp-D-1Nal-D-Trp(Ψ)-Pim;    -   H-Inp-D-2Nal-D-Trp-Thr(Bzl)-NH₂;    -   H-Inp-D-2Nal-D-Trp-Pff-NH₂;    -   H-Inp-D-2Nal-D-Trp(Ψ)-Pim;    -   H-Inp-D-Trp-D-2Nal(Ψ)-Pim;    -   H-Inp-D-2Nal-D-Trp-Taz-NH₂;    -   H-Inp-D-2Nal-D-Dip-Phe-NH₂;    -   H-Inp-D-2Nal-D-Trp-3Pal-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-Taz-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-Phe-Apc-NH₂;    -   H-Inp-D-Bal-D-Trp-Taz-Apc-NH₂;    -   H-Apc-D-Bal-D-Trp-Phe-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-2Thi-Lys-NH₂;    -   H-Apc-D-Bal-D-Bal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-Bal-D-1Nal-Phe-Lys-NH₂;    -   H-Apc-D-Bal-D-2Nal-Phe-Lys-NH₂;    -   H-Inp-D-1Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-1Nal-D-Trp-Taz-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Phe-Apc-NH₂;    -   H-Inp-D-1Nal-D-Trp-Taz-Apc-NH₂;    -   H-Inp-D-1Nal-D-Trp-Phe-Apc-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-1Nal-D-1Nal-Phe-Lys-NH₂;    -   H-Apc-D-2Nal-D-Trp-Phe-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-Phe-Apc-NH₂;    -   H-Apc-D-Bal-D-Trp-Taz-Apc-NH₂;    -   H-Apc-D-Bal-D-1Nal-Phe-Apc-NH₂;    -   H-Apc-D-Bal-D2Nal-Phe-Apc-NH₂;    -   H-Apc-D-1Nal-Trp-Taz-Apc-NH₂;    -   H-Apc-D-1Nal-D-Trp-Phe-Apc-NH₂;    -   H-Apc-D-1Nal-D-1Nal-Phe-Apc-NH₂;    -   H-Apc-D-1Nal-D-2Nal-Phe-Apc-NH₂;    -   H-Inp-D-Bal-D-Trp(Ψ)-Pim;    -   H-Apc-D-Bal-D-Trp-Phe-NH₂;    -   H-Apc-D-Bal-D-Trp-2Thi-NH₂;    -   H-Apc-D-Bal-D-Trp-Taz-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Thi-NH₂;    -   H-Apc-D-1Nal-D-Trp-Taz-NH₂;    -   H-Apc-D-2Nal-D-Trp-2Thi-NH₂;    -   H-Apc-D-2Nal-D-Trp-Taz-NH₂; or    -   H-Aib-D-Ser(Bzl)-D-Trp(Ψ)-Pim;        or a pharmaceutically acceptable salt thereof.

A preferred Group 2B compound, termed a Group 2B-1 compound, is acompound according b the formula:

-   -   H-Inp-D-1Nal-D-Trp-3Pal-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-4Pal-Lys-NH₂;    -   H-Inp-D-Bip-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Thr(Bzl)-Lys-NH₂;

A more preferred Group 2B-1 compound, termed a Group 2B-1b compound, isa compound according to the formula:

-   -   H-Inp-D-2Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-Bal-Trp-Phe-Lys-NH₂;    -   H-Inp-D-1Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-1Nal-D-Trp-Phe-Apc-NH₂;    -   H-Inp-D-Bal-D-Trp-Phe-Apc-NH₂;    -   H-Apc-D-2Nal-D-Trp-Phe-Lys-NH₂;    -   H-Apc-D-1 Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-2Thi-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-Phe-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-Phe-Apc-NH₂; or    -   H-Apc-D-2Nal-D-Trp-2Thi-NH₂;        or a pharmaceutically acceptable salt thereof.

A still more preferred Group 2B1 compound, termed a Group 2B-1ccompound, is a compound according to the formula:

-   -   H-Inp-D-2Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Inp-D-Bal-D-Trp-Phe-Apc-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Thi-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-Taz-Lys-NH₂;        or a pharmaceutically acceptable salt thereof.

A particularly preferred Group 2B-1c compound is a compound according tothe formula:

-   -   H-Inp-D-Bal-D-Trp-Phe-Apc-NH₂;        or a pharmaceutically acceptable salt thereof.

Another still more preferred Group 2B-1 compound, termed a Group 2B1dcompound, is a compound according to the formula:

-   -   H-Inp-D-Bal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-Bal-D-Trp-Taz-Lys-NH₂;    -   H-Apc-D-1Nal-D-Trp-Phe-Apc-NH₂; or        or a pharmaceutically acceptable salt thereof.

Another preferred Group 2B compound, termed a Group 2B-2 compound, is acompound according to the formula:

-   -   H-Inp-D-2Nal-D-Trp-Om-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Pff-Lys-NH₂;    -   H-Inp-D-Dip-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-Bpa-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-2Nal-D-Bpa-Phe-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Pff-NH₂;    -   H-Inp-D-2Nal-D-Dip-Phe-NH₂;    -   H-Inp-D-Trp-D-2Nal(Ψ)-Pim;    -   H-Inp-D-2Nal-D-Trp(Ψ)-Pim;    -   H-Inp-D-1 Na-D-Trp(Ψ)-Pim;    -   H-Inp-D-Bal-D-Trp(Ψ)-Pim; or    -   H-Aib-D-Ser(Bzl)-D-Trp(Ψ)-Pim;        or a pharmaceutically acceptable salt thereof.

A preferred Group 2B-2 compound, termed a Group 2B2a compound, is acompound according to the formula:

-   -   H-Inp-D-2Nal-D-Trp-Pff-Lys-NH₂;    -   H-Inp-D-Dip-D-Trp-Phe-Lys-NH₂;    -   H-Inp-D-2Nal-D-Trp-Pff-NH₂;    -   H-Inp-D-1Na-D-Trp(Ψ)-Pim; or    -   H-Inp-D-Bal-D-Trp(Ψ)-Pim;        or a pharmaceutically acceptable salt thereof.

Another preferred Group 2 compound, termed a Group 2C compound, is acompound according to the formula:

-   -   H-Inp-D-2Nal-D-Trp-3Pal-NH₂;    -   H-Inp-D-2Nal-D-Trp-4-Pal-NH₂;    -   H-Inp-D-1Nal-D-Trp-3Pal-NH₂;    -   H-Inp-D-Bip-D-Trp-Phe-NH₂;    -   H-Inp-D-2Nal-D-Trp-2Thi-NH₂;    -   H-Inp-D-2Nal-D-Trp-3Thi-NH₂;    -   H-Inp-D-Bip-D-Trp-Phe-NH₂;    -   H-Inp-D-Bal-D-Trp-Phe-NH₂;    -   H-Inp-D-2Nal-D-Bal-Phe-NH₂;    -   H-Inp-D-1Nal-D-Trp-2Thi-NH₂; or    -   H-Apc-D-1Nal-D-Trp-Phe-NH₂;        or a pharmaceutically acceptable salt thereof.

A preferred Group 2C compound, termed a Group 2C-1 compound, is acompound according to the formula:

-   -   H-Inp-D-2Nal-D-Trp-2Thi-NH₂;    -   H-Inp-D-Bal-D-Trp-Phe-NH₂;    -   H-Inp-D-1Nal-D-Trp-2Thi-NH₂; or    -   H-Apc-D-1Nal-D-Trp-Phe-NH₂;        or a pharmaceutically acceptable salt thereof.

A particularly preferred compound of the invention, termed a Group 3compound, is a compound according to the formula:

-   -   H-Inp-D-1Nal-D-Trp-2Thi-Apc-NH₂;    -   H-Inp-D-Bal-D-Trp-2Thi-Apc-NH₂;    -   H-Apc-D-1Nal-D-Trp-2Thi-Apc-NH₂;    -   H-Apc-D-Bal-D-Trp-2Thi-Apc-NH₂; or    -   H-Apc-D-1Nal-D-Trp-Phe-Lys-NH₂;        or a pharmaceutically acceptable salt thereof.

In another aspect the invention features a method of determining acompound's ability to bind to a GHS receptor, said method comprising thestep of measuring the ability of a compound to affect binding of acompound according to formula (I) or according to any one of Groups 1,1A, 2, 2A, 2B, 2B-1, 2B-1a, 2B-1b, 2B-1c, 2B-1d, 2B-2, 2B2a, 2C, or 2C-1to said receptor, to a fragment of said receptor, to a polypeptidecomprising said fragment of said receptor, or to a derivative of saidpolypeptide.

In another aspect the invention features a method for achieving abeneficial affect in a subject comprising, said method comprising thestep of administering to said subject an effective amount of a compoundaccording to formula (I), Group 1, Group 1A, Group 2, Group 2A Group 2B,Group 2B-1, Group 2B-1a, Group 2B-1b, Group 2B-1c, Group 2B-1d, Group2B-2, Group 2B-2a, Group 2C, or Group 2C-1, or a pharmaceuticallyacceptable salt thereof, wherein said effective amount is effective forproducing a beneficial effect In helping to treat (e.g., cure or reducethe severity) or prevent (e.g., reduce the likelihood of onset orseverity) a disease or disorder.

In another aspect the invention features a method for stimulating growthhormone secretion in a subject in need of such stimulation, comprisingthe step of administering to a subject an effective amount of a ghrelinagonist according to formula (I), Group 1, Group 1A, Group 2, Group 2A,Group 2B, Group 2B-1, Group 2B-1a, Group 2B-1b, Group 2B-1c, Group2B-1d, Group 2B-2, Group 2B-2a, Group 2C, or Group 2C-1, or apharmaceutically acceptable salt thereof, wherein said effective amountis at least an amount sufficient to produce a detectable increase ingrowth hormone secretion and, preferably, is an amount sufficient toachieve a beneficial affect in a patent.

In one embodiment of the immediately foregoing aspect said stimulationof growth hormone secretion is indicated for treatment of a growthhormone deficient state, for increasing muscle mass, for increasing bonedensity, for sexual dysfunction In males or females, for facilitating aweight gain, for facilitating maintenance of weight, for facilitatingmaintenance of physical functioning, for facilitating recovery ofphysical function, and/or facilitating appetite increase. Preferablysaid facilitating weight gain, facilitating maintenance in weight,and/or facilitating appetite increase is indicated in a patient having adisease or disorder, or under going a treatment, accompanied by weightloss. More preferably said diseases or disorders accompanied by weightloss include anorexia, bulimia, cancer cachexia, AIDS, (e.g., wasting),cachexia, and wasting in frail elderly. Also preferably said treatmentsaccompanied by weight loss include chemotherapy, radiation therapy,temporary or permanent immobilization, and dialysis.

In another aspect the invention features a method for suppressing growthhormone secretion in a subject in need of such suppression, comprisingthe step of administering to a subject an effective amount of a ghrelinantagonist according to formula (I), Group 1, Group 1A, Group 2, Group2A, Group 2B, Group 2B1, Group 2B-1a, Group 2B1b, Group 2B-1c, Group2B-1d, Group 2B-2, Group 2B-2a, Group 2C, or Group 2C-1, or apharmaceutically acceptable salt thereof, wherein said effective amountis at least an amount sufficient to produce a detectable decrease ingrowth hormone secretion and, preferably, is an amount sufficient toachieve a beneficial affect in a patient.

In one embodiment of the immediately foregoing aspect said suppressionof growth hormone secretion is indicated for the treatment of a diseaseor condition characterized by excessive growth hormone secretion, forfacilitation of weight loss, for facilitation of appetite decrease, forfacilitation of weight maintenance, for treating obesity, for treatingdiabetes, for treating complications of diabetes Including retinopathy,and/or for treating cardiovascular disorders.

In a preferred embodiment of the immediately foregoing aspect excessiveweight Is a contributing factor to a disease or condition includinghypertension, diabetes, dyslipidemia, cardiovascular disease, gallstones, osteoarthritis and cancers. More preferably said facilitation ofweight loss reduces the likelihood of such diseases or conditions. Alsomore preferably said facilitation of weight loss comprises at least partof a treatment for such diseases or conditions.

A method of eliciting a ghrelin agonist effect in a subject comprisingthe step of administering to a subject an effective amount of one ormore of a ghrelin agonist according to formula (I), Group 1, Group 1A,Group 2, Group 2A, Group 2B, Group 2B-1, Group 2B-1a, Group 2B-1 b,Group 2B-1c, Group 2B-1d, Group 2B-2, Group 2B-2a, Group 2C, or Group2C-1, or a pharmaceutically acceptable salt thereof, wherein saideffective amount is at least an amount sufficient to produce adetectable increase in growth hormone secretion and, preferably, is anamount sufficient to achieve a beneficial affect in a patent.

In another aspect the invention features a method of eliciting a ghrelinantagonist effect in a subject comprising the step of administering to asubject an effective amount of one or more of a ghrelin antagonistaccording to formula (I), Group 1, Group 1A, Group 2, Group 2A, Group2B, Group 2B-1, Group 2B-1 a, Group 2B-1b, Group 2B-1c, Group 2B-1d,Group 2B-2, Group 2B-2a, Group 2C, or Group 2C-1, or a pharmaceuticallyacceptable salt thereof, wherein said effective amount is at least anamount sufficient to produce a detectable decrease in growth hormonesecretion and, preferably, is an amount sufficient to achieve abeneficial affect In a patient.

Compounds of the invention are active at the GHS receptor. The compoundscan bind to the receptor, and preferably, stimulate receptor activity.Thus a compound of the invention Is useful as a functional ghrelinanalog, both as a research tool and/or as a therapeutic agent.

Research tool applications generally involve the use of a compound ofthe Invention and the presence of a GHS receptor or fragment thereof.The GHS receptor can be present in different environments such as amammalian subject a whole cell, or a cell membrane fragment Examples ofresearch tool applications include screening for compounds active at theGHS receptor, determining the presence of the GHS receptor in a sampleor preparation, and examining the role or effect of ghrelin.

One aspect of the present invention features a method of screening forghrelin agonists and/or for ghrelin antagonists. Screening for ghrelinagonists can be performed, for example, by using a compound according toformula (I), Group 1, Group 1A, Group 2, Group 2A, Group 2B, Group 2B-1,Group 2B-1a, Group 2B-1b, Group 2B-1c, Group 2B-1d, Group 2B-2, Group2B-2a, Group 2C, or Group 2C-1, or a pharmaceutically acceptable saltthereof, in a competition experiment with test compounds. Screening forghrelin antagonists can be performed, for example, by using a compoundaccording to formula (I), Group 1, Group 1A, Group 2, Group 2A, Group2B, Group 2B-1, Group 2B-1a, Group 2B-1b, Group 2B-1c, Group 2B-1d,Group 2B-2, Group 2B-2a, Group 2C, or Group 2C-1, or a pharmaceuticallyacceptable salt thereof, to produce GHS receptor activity and thenmeasuring the ability of a test compound to alter GHS receptor activity.

Another aspect of the present invention features a method of screeningfor a compound able to bind to a GHS receptor. The method comprises thestep of measuring the ability of a test compound to affect the bindingof a compound according to formula (I), Group 1, Group 1A, Group 2,Group 2A, Group 2B, Group 2B-1, Group 2B-1a, Group 2B-1b, Group 2B-1c,Group 2B-1d, Group 2B-2, Group 2B-2a, Group 2C, or Group 2C-1, or apharmaceutically acceptable salt thereof, to either the receptor, afragment of the receptor comprising a ghrelin binding site, apolypeptide comprising the fragment, or a derivative of the polypeptide.

Ghrelin agonists can be used to achieve a beneficial effect in asubject. For example, ghrelin induces growth hormone release fromprimary-culture pituitary cells in a dose-dependent manner withoutstimulating the release of the other pituitary hormones. Injectedintravenously into anaesthetized rats, ghrelin stimulated pulsatilerelease of growth hormone. (Kojima et al., Nature 1999, 402, 656-660.)Thus a non-exclusive list of examples wherein such a beneficial effectmay be indicated would include: treating a growth hormone deficientstate, increasing muscle mass, increasing bone density, treating sexualdysfunction in males or females, facilitating a weight gain,facilitating maintenance of weight, facilitating maintenance of physicalfunctioning, facilitating recovery of physical function, and/orfacilitating appetite increase. Facilitating a weight gain, maintenancein weight, or appetite increase is particularly useful for a subjecthaving a disease or disorder, or undergoing a treatment, accompanied byweight loss. Diseases or disorders accompanied by weight loss include,e.g., anorexia, bulimia, cancer cachexia, AIDS, (e.g., wasting),cachexia, wasting In frail elderly, and the like. Treatments accompaniedby weight loss include, e.g., chemotherapy, radiation therapy, temporaryor permanent immobilization, dialysis, and the like.

Thus another aspect of the present invention features a method forachieving a beneficial affect In a subject, said method comprising thestep of administering to said subject an effective amount of one or moreof a compound according to formula (I), Group 1, Group 1A, Group 2,Group 2A, Group 2B, Group 2B-1, Group 2B-1a, Group 2B-1b, Group 2B-1c,Group 2B-1d, Group 2B-2, Group 2B-2a, Group 2C, or Group 2C1, or apharmaceutically acceptable salt thereof, wherein said effective amountis effective for producing a beneficial effect in helping to treat(e.g., cure or reduce the severity of) or prevent (e.g., reduce thelikelihood of onset or severity of) a disease or disorder.

In a preferred embodiment of the immediately preceding method saidbeneficial affect comprises stimulating growth hormone secretion in asubject In need of such stimulation, comprising the step ofadministering to a subject an effective amount of one or more of acompound according to formula (I), Group 1, Group 1A, Group 2, Group 2A,Group 2B, Group 2B-1, Group 2B-1a, Group 2B-1b, Group 2B-1c, Group2B-1d, Group 2B-2, Group 2B-2a, Group 2C, or Group 2C-1, or apharmaceutically acceptable salt thereof, wherein said effective amountis at least an amount sufficient to produce a detectable increase ingrowth hormone secretion and, preferably, is an amount sufficient toachieve a beneficial affect in a patient.

In a more preferred embodiment of the immediately preceding method saidstimulation of growth hormone secretion is Indicated for treatment of agrowth hormone deficient state, for increasing muscle mass, forincreasing bone density, for sexual dysfunction in males or females, forfacilitating a weight gain, for facilitating maintenance of weight, forfacilitating maintenance of physical functioning, for facilitatingrecovery of physical function, and/or facilitating appetite increase.

In another preferred embodiment of the immediately preceding method saidfacilitating weight gain, facilitating maintenance in weight, and/orfacilitating appetite increase is indicated in a patient having adisease or disorder, or under going a treatment, accompanied by weightloss. More preferably said diseases or disorders accompanied by weightloss include anorexia, bulimia, cancer cachexia, AIDS, (e.g., wasting),cachexia, and wasting in frail elderly.

In another more preferred embodiment of the Immediately preceding methodsaid treatments accompanied by weight loss Include chemotherapy,radiation therapy, temporary or permanent immobilization, and dialysis.

Ghrelin antagonists can also be used to achieve a beneficial effect in apatient. For example, a ghrelin antagonist can be used to facilitateweight loss, facilitate appetite decrease, facilitate weightmaintenance, treat obesity, treat diabetes, treat complications ofdiabetes Including retinopathy, and/or treat cardiovascular disorders.Excessive weight is a contributing factor to different diseasesincluding hypertension, diabetes, dyslipidemias, cardiovascular disease,gall stones, osteoarthritis and certain forms of cancers. Bringing abouta weight loss can be used, for example, to reduce the likelihood of suchdiseases and as part of a treatment for such diseases.

Compounds of the invention may also antagonize the effects of ghrelin invitro and in vivo. Thus yet another aspect of the present inventionfeatures a method for suppressing growth hormone secretion in a subjectin need of such suppression, comprising the step of administering to asubject an effective amount of one or more of a compound according toformula (I), Group 1, Group 1A, Group 2, Group 2A, Group 2B, Group 2B-1,Group 2B1a, Group 2B-1b, Group 2B-1c, Group 2B-1d, Group 2B-2, Group2B-2a, Group 2C, or Group 2C-1, or a pharmaceutically acceptable saltthereof, wherein said effective amount Is at least an amount sufficientto produce a detectable decrease in growth hormone secretion and,preferably, is an amount sufficient to achieve a beneficial affect in apatient.

In a preferred embodiment of the immediately preceding method saidsuppression of growth hormone secretion Is Indicated for the treatmentof a disease or condition characterized by excessive growth hormonesecretion, for facilitation of weight loss, for facilitation of appetitedecrease, for facilitation of weight maintenance, for treating obesity,for treating diabetes, for treating complications of diabetes includingretinopathy, and/or for treating cardiovascular disorders.

In a more preferred embodiment of the immediately preceding methodexcessive weight is a contributing factor to a disease or conditionincluding hypertension, diabetes, dyslipidemia, cardiovascular disease,gall stones, osteoarthritis and cancers.

In another more preferred embodiment of the immediately preceding methodsaid facilitation of weight loss reduces the likelihood of such diseasesor conditions and/or said facilitation of weight loss comprises at leastpart of a treatment for such diseases or conditions.

As is also appreciated by those of skill in the art, ghrelin andagonists thereof may also be used to achieve beneficial cardiovasculareffects. (Nagaya, et al., Regul Pept. 2003 Jul. 15: 114 (2-3): 71-77.)For example, it is known that ghrelin inhibits apoptosis ofcardiomyocytes and endothelial cells in vitro, that repeatedadministration of ghrelin improves cardiac structure and function andattenuates the development of cardiac cachexia in rats wit heartfailure, and that ghrelin decreases systemic vascular resistance andIncreases cardiac output in human patients with heart failure. (Id.)Thus it has been recognized that ghrelin and ghrelin agonists representpotential therapeutics for the treatment of severe chronic heartfailure.

In a particularly preferred embodiment of each of the methods of using aghrelin agonist described herein the ghrelin agonist is a compoundaccording to the formula:

-   -   H-Inp-D-Bal-D-Trp-Phe-Apc-NH₂;        or a pharmaceutically acceptable salt thereof.

A compound or compounds of the invention can be administered to asubject. A “subject” refers to a mammalian or non-mammalian animalincluding, for example and without limitation, a human, a rat a mouse,or a farm animal. Reference to subject does not necessarily indicate thepresence of a disease or disorder. Thus the term subject furtherincludes, for example, a mammalian or non-mammalian animal being dosedwith a ghrelin analog as part of an experiment, a mammalian ornon-mammalian animal being treated to help alleviate a disease ordisorder, and a mammalian or non-mammalian animal being treatedprophylactically to retard or prevent the onset of a disease ordisorder.

Other features and advantages of the present invention are apparent fromthe additional descriptions provided herein including the differentexamples. The provided examples illustrate different components andmethodology useful in practicing the present invention. The examples donot limit the claimed invention. Based on the present disclosure theskilled artisan can identify and employ other components and methodologyuseful for practicing the present invention.

Unless otherwise stated, those amino adds with a chiral center areprovided in the L-enantiomer. Reference to “a derivative thereof” refersto a modified amino acid such as the corresponding D-amino acid, aN-alkyl-amino acid, a β-amino acid, or a labeled amino acid.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features peptidyl analogs active at the GHSreceptor. Human ghrelin is a 28 amino acid modified peptide where aserine hydroxy group is esterified by n-octanoic acid. (Kojima et al.,Nature 1999, 402, 656-660, and Kojima, (Abstract), Third InternationalSymposium on Growth Hormone Secretagogues, Keystone, Colo., USA 2000,Feb. 17-19.)

Certain amino adds present in compounds of the invention are representedherein as follows:

-   -   A3c 1-amino-1-cyclopropanecarboxylic acid    -   A4c 1-amino-1-cyclobutanecarboxylic acid    -   A5c 1-amino-1-cyclopentanecarboxylic acid    -   A6c 1-amino-1-cyclohexanecarboxylic acid    -   Abu α-aminobutyric acid    -   Acc 1-amino-1-cyclo(C₃-C₉)alkyl carboxylic acid    -   Act 4-amino-4-carboxytetrahydropyran, i.e.,:

-   -   Aib α-aminoisobutyric acid    -   Ala or A alanine    -   β-Ala beta-alanine    -   Apc amino piperidinylcarboxylic acid, i.e.:

-   -   Arg or R arginine    -   hArg homoarginine    -   Asn or N asparagine    -   Asp or D aspartic acid    -   Bal 3-Benzothienylalanine, i.e.:

-   -   Bip 4,4′-Biphenylalanine, i.e.:

-   -   Bpa 4-Benzoylphenylalanine, i.e.:

-   -   Cha β-cyclohexyalanine;    -   Cys or C cysteine;    -   Dab 2,4-diaminobutyric acid, (α,γ-Diaminobutyric acid);    -   Dap 2,3-diaminopropionic acid, (α,β-Diaminopropionic add);    -   Dip β,β-Diphenylalanine, i.e.:

-   -   Dhp 3,4-dehydroproline    -   Dmt 5,5-dimethylthiazolidine-4-carboxylic acid    -   2Fua β-(2-furyl)-alanine, i.e.:

-   -   Gin or Q glutamine    -   Glu or E glutamic acid    -   Gly or G glycine    -   His or H histidine    -   3Hyp trans-3-hydroxy-L-proline, i.e., (2S,        3S)-3-hydroxypyrrolidine-2-carboxylic acid;    -   4Hyp 4-hydroxyproline, i.e., (2S,        4R)-4-hydroxypyrrolidine-2-carboxylic acid;    -   Ile or I isoleucine    -   Inc indoline-2-carboxylic acid    -   Inp isonipecotic acid, i.e.:

-   -   Ktp 4-ketoproline    -   Leu or L leucine    -   hLeu homoleucine    -   Lys or K lysine    -   Met or M methionine    -   1Nal β-(1-Naphthyl)alanine;    -   2Nal β-(2-Naphthyl)alanine;    -   Nle norleucine    -   Nva norvaline    -   Oic octahydroindole-2-carboxylic acid    -   Orn ornithine    -   2Pal β-(2-Pyridyl)-alanine, i.e.,

-   -   3Pal β-(3-Pyridyl)-alanine, i.e.:

-   -   4Pal β-(4-Pyridyl)-alanine, i.e.:

-   -   Pff pentafluorophenylalanine, i.e.

-   -   Phe or F phenylalanine    -   hPhe homophenylalanine    -   Pim 2′-(4-Phenyl)imidazolyl, i.e.:

-   -   Pip pipecolic acid    -   Pro or P proline    -   Ser or S serine    -   Taz β-(4-thiazolyl)alanine, i.e.,

-   -   2Thi β-(2-thienyl)alanine, i.e.:

-   -   3Thi β-(3-thienyl)alanine, i.e.:

-   -   Thr or T threonine    -   Thz thiazolidine-4-carboxylic acid    -   Tic 1,2,3,4-tetrahydroisoquinoline-carboxylic acid    -   Tle tert-leucine    -   Trp or W tryptophan    -   Tyr or Y tyrosine    -   Val or V valine

Certain other abbreviations used herein are defined as follows:

-   -   Boc: tert-butyloxycarbonyl    -   Bzl: benzyl    -   DCM: dichloromethane    -   DIC: N,N-diisopropylcarbodiimide    -   DIEA: diisopropylethyl amine    -   Dmab:        4{N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl)-amino}benzyl    -   DMAP: 4-(dimethylamino)pyridine    -   DMF dimethylformamide    -   DNP: 2,4-dinitrophenyl    -   Fmoc: Fluorenylmethyloxycarbonyl    -   HBTU: 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate    -   cHex cyclohexyl    -   HOAT: O-(7-azabenzotriazol-1-yl)1,1,3,3-tetramethyluronium        hexafluorophosphate    -   HOBt 1-hydroxy-benzotriazole    -   HOSu: N-hydroxysuccinimide    -   Mmt 4-methoxytrityl    -   NMP: N-methylpyrrolidone    -   Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl    -   tBu: tert-butyl    -   TIS: triisopropylsilane    -   TOS: tosyl    -   trt trityl    -   TFA: trifluoro acetic acid    -   TFFH: tetramethylfluoroforamidinium hexafluorophosphate    -   Z: benzyloxycarbonyl

Unless otherwise apparent, abbreviations (e.g. Ala) of amino acids inthis disclosure stand for the structure of —NH—C(R)(R′)—CO—, wherein Rand R′ each is, independently, hydrogen or the side chain of an aminoacid (e.g., R=CH₃ and R′=H for Ala), or R and R′ may be joined to form aring system.

“Alkyl” refers to a hydrocarbon group containing one or more carbonatoms, where multiple carbon atoms if present are joined by singlebonds. The alkyl hydrocarbon group may be straight-chain or contain oneor more branches or cyclic groups.

“Substituted alkyl” refers to an alkyl wherein one or more hydrogenatoms of the hydrocarbon group are replaced with one or moresubstituents selected from the group consisting of halogen, (i.e.,fluorine, chlorine, bromine, and iodine), —OH, —CN, —SH, —NH₂, —NHCH₃,—NO₂, —C₁₋₂ alkyl substituted with 1 to 6 halogens, CF₃, —OCH₃, —OCF₃,and —CH₂)₀₋₄—COOH. In different embodiments 1, 2, 3 or 4 substituentsare present. The presence of —(CH₂)₀₋₄—COOH results in the production ofan alkyl acid. Examples of alkyl acids containing, or consisting of,—CH₂)₀₋₄—COOH include 2-norbornane acetic acid, tert-butyric acid and3-cyclopentyl propionic acid.

“Heteroalkyl” refers to an alkyl wherein one of more of the carbon atomsin the hydrocarbon group are replaced with one or more of the followinggroups: amino, amido, —O—, or carbonyl. In different embodiments 1 or 2heteroatoms are present.

“Substituted heteroalkyl” refers to a heteroalkyl wherein one or morehydrogen atoms of the hydrocarbon group are replaced with one or moresubstituents selected from the group consisting of halogen, (i.e.,fluorine, chlorine, bromine, and iodine), —H, —CN, —SH, —NH₂, —NHCH₃,—NO₂, —C₁₋₂ alkyl substituted with 1 to 6 halogens, —CF₃, —OCH₃, —OCF₃,and —CH₂)₀₋₄—COOH. In different embodiments 1, 2, 3 or 4 substituentsare present.

“Alkenyl” refers to a hydrocarbon group made up of two or more carbonswhere one or more carbon-carbon double bonds are present. The alkenylhydrocarbon group may be straight-chain or contain one or more branchesor cyclic groups.

“Substituted alkenyl” refers to an alkenyl wherein one or more hydrogensare replaced with one or more substituents selected from the groupconsisting of halogen (i.e., fluorine, chlorine, bromine, and iodine),—H, —CN, —SH, —NH₂, —NHCH₃, —NO₂, —C₁₋₂ alkyl substituted with 1 to 6halogens, —CF₃, —OCH₃, OCF₃, and —(CH₂)₀₋₄—COOH. In differentembodiments 1, 2, 3 or 4 substituents are present.

“Aryl” refers to an optionally substituted aromatic group with at leastone ring having a conjugated pi-electron system, containing up to twoconjugated or fused ring systems. Aryl includes carbocyclic aryl,heterocyclic aryl and biaryl groups. Preferably, the aryl Is a 5 or 6membered ring. Preferred atoms for a heterocyclic aryl are one or moresulfur, oxygen, and/or nitrogen. Examples of aryl include phenyl,1-naphthyl, 2-naphthyl, indole, quinoline, 2-imidazole, and9-anthracene. Aryl substituents are selected from the group consistingof —C₁₋₄ alkyl, —C₁₋₄ alkoxy, halogen (i.e., fluorine, chlorine,bromine, and iodine), —OH, —CN, OH, NH₂, —NO₂, —C₁₋₂ alkyl substitutedwith 1 to 5 halogens, —CF₃, —OCF₃, and —(CH₂)₀₋₄—COOH. In differentembodiments the aryl contains 0, 1, 2, 3, or 4 substituents.

“Alkylaryl” refers to an “alkyl” joined to an “aryl”.

When a non-amino acid imidazole moiety, (e.g., Pim, defined above), ispresent at the C-terminus of a compound of the invention It Isunderstood that the imidazole moiety Is attached to the adjacent aminoacid via a pseudo-peptide bond, wherein a bond is formed between theposition 2 carbon of the imidazole ring and the alpha carbon of theamino acid. For example, in the case where the adjacent amino acid isD-tryptophan (D-Trp) and the imidazole moiety is Pim, the C-terminus ofthe peptide would appear as follows:

For clarity, in the written formula for such a compound the presence ofthis bond is indicated by the Greek letter “Ψ” alone in parentheses. Forexample, the written formula H-Inp-D-Trp-D-2Nal(Ψ)-Pim denotes thestructure:

The present invention includes diastereomers as well as their racemicand resolved enantiomerically pure forms. Ghrelin analogs can containD-amino acids, L-amino acids or a combination thereof. Preferably, aminoacids present in a ghrelin analog are the L-enantiomers.

Preferred derivatives of analogs of the invention comprise D-amino adds,N-alkyl-amino acids, β-amino acids, and/or one or more labeled aminoadds (including a labeled version of a D-amino acid, a N-alkyl-aminoacids, or a β-amino add). A labeled derivative indicates the alterationof an amino acid or amino acid derivative with a detectable label.Examples of detectable labels include luminescent, enzymatic, andradioactive label. Both the type of label and the position of the labelcan effect analog activity. Labels should be selected and positioned soas not to substantially after the activity of the ghrelin analog at theGHS receptor. The effect of a particular label and position on ghrelinactivity can be determined using assays measuring ghrelin activityand/or binding.

A protecting group covalently joined to the C-terminal carboxy groupreduces the reactivity of the carboxy terminus under in vivo conditions.The carboxy terminus protecting group is preferably attached to theα-carbonyl group of the last amino acid. Preferred carboxy terminusprotecting groups include amide, methylamide, and ethylamide.

EXAMPLES

Examples are provided below to further illustrate different features ofthe present invention. The examples also illustrate useful methodologyfor practicing the invention. These examples do not limit the claimedinvention.

Synthesis

The compounds of the invention can be produced using the techniquesdisclosed in the examples herein as well as techniques that are wellknown in the art For example, a polypeptide region of a GHRP analog canbe chemically or biochemically synthesized and modified. Examples oftechniques for biochemical synthesis involving the introduction of anucleic acid into a cell and expression of nucleic acids are provided inAusubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998,and Sambrook et al., in Molecular Cloning, A Laboratory Manual, 2^(nd)Edition, Cold Spring Harbor Laboratory Press, 1989. Techniques forchemical synthesis of polypeptides are also well known in the art (Seee.g., Vincent in Peptide and Protein Drug Delivery, New York, N.Y.,Dekker, 1990.) For example, the peptides of this invention can beprepared by standard solid phase peptide synthesis. (See, e.g., Stewart,J. M., et al., Solid Phase Synthesis (Pierce Chemical Co., 2d ed.1984).)

The substituent R¹ of the above formula (I) may be attached to the freeamine of the N-terminal amino acid by standard methods known in the artFor example, alkyl groups, e.g., (C₁-C₃₀)alkyl, may be attached usingreductive alkylation. Hydroxyalkyl groups, e.g., (C₁-C₃₀)hydroxyalkyl,may also be attached using reductive alkylation wherein the free hydroxygroup is protected with a t-butyl ester. Acyl groups, e.g., COE¹, may beattached by coupling the free acid, e.g., E¹COOH, to the free amine ofthe N-terminal amino acid by mixing the completed resin with 3 molarequivalents of both the free acid and diisopropylcarbodiimide inmethylene chloride for about one hour. If the free acid contains a freehydroxy group, e.g., p-hydroxyphenylpropionic acid, then the couplingshould be performed with an additional 3 molar equivalents of HOBT.

Peptides of the invention also can be and were synthesized in a parallelfashion on an ACT 396 Multiple Biomolecular Synthesizer (AdvancedChemTech, Louisville, Ky.), (“synthesizer”), as follows. The synthesizerwas programmed to perform the following reaction cycle: (1) washing withdimethylformamide (DMF), (2) removing Fmoc protecting group with 20%piperidine in DMF for 1×5 min and 1×25 min, (3) washing with DMF, (4)coupling with Fmoc amino acid for 1 h at room temperature in thepresence of diispropylcarbodiimide (DIC) and 1-hydroxybenzotriazole(HOBt), and (5) repeating step 4.

Examples 1-65

Each of the reaction wells contained 0.0675 mmol of Rink Amide MBHAresin (substitution=0.72 mmol/g, Novabiochem, San Diego, Calif.). Thefollowing Fmoc amino acids (Novabiochem, San Diego, Calif.; Chem-ImpexInternational, Wood Dale, Ill.; SyntheTech, Albany, Oreg.; Pharma Core,High Point, N.C.) were used: Fmoc Lys(Boc)-OH, Fmoc-Phe-OH,Fmoc-H-Inp-OH, Fmoc-D-1Nal-OH, Fmoc-D-2Nal-OH, Fmoc-D-Trp(Boc)-OH,Fmoc-3Pal-OH, Fmoc-4Pal-OH, Fmoc-Orn(Boc)-OH, Fmoc-D-Bip-OH,Fmoc-Thr(Bzl)-OH, Fmoc-Pff-OH, Fmoc-2Thi-OH, Fmoc-Taz-OH, Fmoc-D-Dip-OH,Fmoc-Bpa-OH, Fmoc-D-Bal-OH, and Fmoc-Apc(Boc)-OH.

Each of the Fmoc amino acids was dissolved in a 0.3 N solution of HOBtin DMF wherein the concentration of the resulting Fmoc amino acid was0.3 N. A four fold excess (0.27 mmol, 0.9 mL of the 0.3 N solution) ofFmoc amino acid was used for each coupling. DIC (0.27 mmol, 0.6 mL of0.45N DIC solution in DMF) was used as the coupling reagent for eachcoupling. Deprotection was performed by using 20% piperidine in DMF(2×1.5 mL per residue).

The peptides were cleaved from the resin by treating the peptide resinswith 8% triisopropylsilane (TIP) in trifluoroacetic acid (TFA) (1.5 mLper reaction well) at room temperature for 2 h. The resin was removed byfiltration. Each filtrate was diluted to 25 mL with ether in acentrifuge tube. The resulting precipitate in each tube was centrifugedand the solvents were decanted from the precipitate. The precipitate ineach tube was then dissolved In methanol (3 mL) and diluted with water(1 mL). The purification of the crude products was done on areverse-phase preparative HPLC using a column (100×21.20 mm, 5μ) of LUNA5μ C8(2) (Phenomenex, Torrance, Calif.). For each peptide, the columnwas eluted with a linear gradient from 85% A and 15% B to 25% A and 75%B in 15 min with a flow rate of 25 mL/min. A was 0.1% TFA in water and Bwas 0.1% TFA in acetonitrile/water (80/20, v/v). The fractions werechecked by analytical HPLC and those containing the pure product werecombined and lyophilized to dryness.

Yields ranged from 13% to 71% and purity of each of Examples 1-65exceeded 94% based upon analytical HPLC analysis. Electro-sprayionization mass spectrometry (ES-MS) analysis was performed and observedmolecular weights were in agreement with calculated molecular weights.The results are detailed in Table I, below.

Examples 66-69

Examples 66-69 were synthesized according to the following procedure.

1.a. BOC-(D)-Trp-OH (4.0 g, 13.1 mmole) (Novabiochem San Diego, Calif.)in methanol (36 ml) and Cs₂CO₃ (2.14 g, 6.57 mmole) in water (10 ml)were combined and the mixture was swirled until a homogeneous mixturewas obtained. Solvents were removed in vacuo and the residue wasdissolved in DMF (45 ml). 2-bromoacetophenone (2.61 g, 13.1 mmole) inDMF (9 ml) was added to the solution and the solution was stirred for 30min. at room temperature. Cesium bromide was removed by filtration andthe filtrate was concentrated in vacuo. The resulting concentrate wasdissolved in xylenes (45 ml), NH₄OAc (17.1 g) was added, and thesolution was heated at reflux for 1 hr. The cooled solution was washedtwo times with saturated NaHCO₃ solution (45 ml) and then with saturatedNaCl. The resulting organic layer was purified by flash chromatographyto yield 4.1 g (77%) of Intermediate 1A depicted in Scheme 1A,(“Compound 1A”).

1b. Compound 1A (403 mg) was deblocked using a mixture oftrifluoroacetic acid (TFA) (8 ml) dichloromethane (DCM) (8 ml) andtriisopropylsilane (TIPS) (1.4 ml). After mixing for one hour thesolution was concentrated under a stream of nitrogen. The residue wasdissolved in DCM (40 ml), washed two times with a saturated solution ofNaHCO₃ (40 ml), and then dried over Na₂SO₄ to yield a solution of theintermediate product 1B, depicted in Scheme 1B, below.

1c.-f. The forgoing solution of the intermediate product 1B was dividedinto four equal portions and coupled with the pre-activated HOBT estersof FMOC protected amino acids, as summarized in reaction schemes 1C, 1D,1E, and 1F, below. The amino acid used for each of example 66, 67, 68and 69 was as follows:

-   -   Ex. 66: FMOC-D-2Nal-OH (130 mg, 0.30 mmole) (Synthetech Albany,        Oreg.)    -   Ex. 67: FMOC-D-1Nal-OH (130 mg, 0.3 mmole) (Advanced Chemtech        Louisville, Ky.)    -   Ex. 68: FMOC-D-Bal-OH (132 mg, 0.30 mmole) (Chem Impex Wood        Dale, Ill.)    -   Ex. 69: FMOC-DSer(Bzl)-OH (124 mg. 0.30 mmole) (Chem Impex Wood        Dale, Ill.)        Each of the immediately foregoing amino acids was pre-activated        with HOBT (46 mg, 0.30 mmole) and DIC (38 mg, 0.30 mmole) in DCM        (5 ml) for ten minutes before addition to one of the four        portions of the forgoing solution of the intermediate product        1B. The coupling reaction was then allowed to proceed for 30        minutes at room temperature.

1.g-j. The FMOC group is removed from each of the resulting compounds1C, 1D, 1E and 1F by addition of tris(2-aminoethyl)amine (0.9 ml) to therespective reaction mixtures from the previous step and mixing for 30minutes at room temperature. The reaction mixtures containing thedeblocked compounds were then washed three times with 10% pH 5.5phosphate buffer (10 ml).

The resulting free amine solutions were coupled with pre-activated HOBTesters of FMOC or BOC protected amino adds, as follows:

-   -   Ex. 66: FMOC-Inp-OH (105 mg, 0.30 mmole) (Chem Impex Wood Dale,        Ill.)    -   Ex. 67: FMOC-Inp-OH (105 mg, 0.30 mmole)    -   Ex 68: BOC-Inp-OH (68.3 mg, 0.30 mmole) (Bachem Torrance, Calif.    -   Ex. 69: BOC-Aib-OH (60.6 mg, 0.30 mmole) (Bachem Torrance,        Calif.)        Each of the immediately foregoing amino acids was pre-activated        with HOBT (46 mg, 0.30 mmole) and DIC (38 mg, 0.30 mmole) in DCM        (5 ml) for ten minutes before addition to the appropriate        deprotected amine. The coupling reaction was then allowed to        proceed for one hour at room temperature.

Deprotection—Compounds 66-67. The FMOC group was removed from theresulting FMOC-protected compounds by addition oftris(2-aminoethyl)amine (0.9 ml) and mixing for 30 minutes. Thedeblocked compounds were washed three times with 10% pH 5.5 phosphatebuffer (10 ml) and the crude products were collected as a precipitate.

Deprotection—Compounds 68-69. The BOC-protected compounds were purifiedby flash chromatography and then deblocked for one hour with TIPS (0.50ml), TFA (0.50 ml), in DCM (2.75 ml). The crude products were thenconcentrated and dried under vacuum.

Purification by HPLC afforded the products in 5% and 29% yields for thecompounds of examples 66 and 67, respectively, and 15% and 43% for thecompounds of examples 68 and 69, respectively.

The foregoing deprotection, coupling, and deprotection steps aresummarized in reaction schemes 1G, 1H, 1I and 1J, below

Example 70 H-Inp-D-Trp-D-2Nal(Ψ)-Pim

Compound 70 was synthesized according b the following procedure.

2.a.1 and 2.a.2.: Compound 2A was made in an analogous manner as wasCompound 1A, using BOCD-2Nal-OH and 2-bromoacetophenone as startingmaterials.

Steps 2.a.1. and 2.a2. are summarized in Scheme 2A, below.

2.b.1. Compound 2A (100 mg, 0.242 mmole) was deblocked in TFA (2 ml) andDCM (2 ml) for one hour. Volatiles were then removed under a stream ofnitrogen and the residue was dissolved in DCM (10 ml). The resultingsolution washed three times with saturated NaHCO₃ (10 ml) to yield asolution of Compound 2A in free amine form.

2.b.2. The active ester of FMOC-D-Trp-(BOC)-OH (153 mg, 0.290 mmole) waspreformed with N-hydroxysuccinimide (HOSu; 33 mg, 0.290 mmole) and DIC(37 mg, 0.290 mmole) in DCM (1.5 ml). After one hour diisopropylurea wasremoved by filtration and the filtrate was added to the Compound 2A(free amine) solution. The resulting solution was diluted with DCM to 4ml and the coupling reaction allowed to proceed for 30 minutes.

Steps 2.b.1. and 2.b2. are summarized in Scheme 2B, below.

2.c.1 Compound 2B was deblocked by addition of tris(2-aminoethyl)amine(TAEA) (0.9 ml) to the immediately foregoing coupling reaction solutionand mixing ford 30 minutes at room temperature. The reaction solutionwas then washed three times with saturated NaCl solution (10 ml)followed by three times with 10% pH 5.5 phosphate buffer (10 ml) toyield a solution of Compound 2B In free amine form.

2.c.2. The active ester of BOC-Inp-OH (66.5 mg, 0.290 mmole) waspreformed with HOSu (33 mg, 0.290 mmole) and DIC (37 mg 0.290 mmole) inDCM (1.5 ml). After one hour diisopropylurea was removed by filtrationand the filtrate was added to the Compound 2B (free amine) solution. Theresulting solution was diluted with DCM to 4 ml and the couplingreaction was allowed to proceed for 12 hours.

The reaction mixture was then washed three times with 10% pH 5.5phosphate buffer (10 ml) and dried over Na₂SO₄. Solvent was removedunder vacuum and the concentrate was purified by flash chromatography.

2.c.3. The intermediate was deblocked using TFA (2.75 ml) and TIPS (0.5ml) in DCM (2.75 ml) for 30 minutes. Volatiles were removed from thereaction mixture under a stream of nitrogen and the residue wastriturated with ether (15 ml). After centrifugation the ether wasdecanted and the resulting solid was subjected to HPLC to yield purifiedCompound 70 in 39% yield.

Steps 2.c.1. and 2.c.2. and 2.c.3. are summarized in Scheme 2C, below.

Other peptides of the invention can be prepared by a person of ordinaryskill in the art using synthetic procedures analogous to those disclosedgenerally hereinabove and/or to those disclosed specifically in theforegoing examples, as were the Compounds depicted In Table 1.

TABLE 1 Mol. Wt. Ex. Mol. Wt. (MS- No. Purity Sequence (Calc.) ES) (%) 1H-Inp-D-1Nal-D-Trp-3Pal-Lys-NH₂ 787.96 787.4 96 2H-Inp-D-2Nal-D-Trp-4Pal-Lys-NH₂ 787.96 787.4 99 3H-Inp-D-2Nal-D-Trp-Orn-Lys-NH₂ 753.94 753.4 98 4H-Inp-D-Bip-D-Trp-Phe-Lys-NH₂ 813.01 812.4 99 5H-Inp-D-2Nal-D-Trp-Thr(Bzl)-Lys-NH₂ 831.03 830.4 98 6H-Inp-D-2Nal-D-Trp-Pff-Lys-NH₂ 876.92 876.3 98 7H-Inp-D-2Nal-D-Trp-Thi-Lys-NH₂ 793.00 792.4 98 8H-Inp-D-2Nal-D-Trp-Taz-Lys-NH₂ 793.99 793.4 97 9H-Inp-D-Dip-D-Trp-Phe-Lys-NH₂ 813.01 812.4 98 10H-Inp-D-Bpa-D-Trp-Phe-Lys-NH₂ 841.02 840.4 95 11H-Inp-D-2Nal-D-Bpa-Phe-Lys-NH₂ 852.04 851.3 99 12H-Inp-D-2Nal-D-Trp-3Pal-NH₂ 659.79 659.3 99 13H-Inp-D-2Nal-D-Trp-4Pal-NH₂ 659.79 659.3 98 14H-Inp-D-1Nal-D-Trp-3Pal-NH₂ 659.79 659.3 98 15 H-Inp-D-Bip-D-Trp-Phe-NH₂684.84 684.3 99 16 H-Inp-D-2Nal-D-Trp-Thr(Bzl)-NH₂ 702.85 702.3 99 17H-Inp-D-2Nal-D-Trp-Pff-NH₂ 748.75 748.2 99 18H-Inp-D-2Nal-D-Trp-2Thi-NH₂ 664.83 664.2 99 19H-Inp-D-2Nal-D-Trp-Taz-NH₂ 665.82 665.3 98 20 H-Inp-D-Dip-D-Trp-Phe-NH₂684.84 684.3 98 21 H-Inp-D-2Nal-D-Dip-Phe-NH₂ 695.86 695.3 99 22H-Inp-D-Bal-D-Trp-Phe-NH₂ 664.83 664.3 97 23 H-Inp-D-2Nal-D-Bal-Phe-NH₂675.85 675.2 99 24 H-Inp-D-2Nal-D-Trp-3Pal-Lys-NH₂ 787.96 787.5 97 25H-Inp-D-Bal-D-Trp-2Thi-Lys-NH₂ 799.03 798.4 99 26H-Inp-D-Bal-D-Trp-Phe-Lys-NH₂ 793.00 792.4 99 27H-Inp-D-1Nal-D-Trp-2Thi-Lys-NH₂ 793.00 792.4 99 28H-Inp-D-2Nal-D-Trp-Phe-Apc-NH₂ 784.96 784.4 98 29H-Inp-D-1Nal-D-Trp-Phe-Apc-NH₂ 784.96 784.4 98 30H-Inp-D-Bal-D-Trp-Phe-Apc-NH₂ 790.99 790.4 97 31H-Apc-D-2Nal-D-Trp-Phe-Lys-NH₂ 801.99 801.4 98 32H-Apc-D-1Nal-D-Trp-2Thi-Lys-NH₂ 808.02 807.4 99 33H-Inp-D-1Nal-D-Trp-2Thi-NH₂ 664.83 664.2 98 34H-Apc-D-1Nal-D-Trp-Phe-NH₂ 673.81 673.3 99 35H-Inp-D-1Nal-D-Trp-Taz-Lys-NH₂ 793.99 793.5 99 36H-Inp-D-Bal-D-Trp-Taz-Lys-NH₂ 800.02 799.4 99 37H-Apc-D-1Nal-D-Trp-Taz-Lys-NH₂ 809.00 808.5 99 38H-Apc-D-Bal-D-Trp-Taz-Lys-NH₂ 815.03 814.4 99 39H-Apc-D-Bal-D-Trp-2Thi-Lys-NH₂ 814.04 813.4 98 40H-Inp-D-1Nal-D-Trp-2Thi-Apc-NH₂ 790.99 790.5 97 41H-Inp-D-Bal-D-Trp-2Thi-Apc-NH₂ 797.01 796.4 97 42H-Apc-D-1Nal-D-Trp-2Thi-Apc-NH₂ 806.00 805.5 97 43H-Apc-D-Bal-D-Trp-2Thi-Apc-NH₂ 812.03 811.4 98 44H-Apc-D-1Nal-D-Trp-Phe-Lys-NH₂ 801.99 801.5 98 45H-Apc-D-Bal-D-Trp-Phe-Lys-NH₂ 808.02 807.5 99 46H-Apc-D-1Nal-D-Trp-Phe-Apc-NH₂ 799.97 799.5 98 47H-Apc-D-Bal-D-Trp-Phe-Apc-NH₂ 806.00 805.5 98 48H-Apc-D-1Nal-D-1Nal-Phe-Apc-NH₂ 811.00 810.5 95 49H-Apc-D-1Nal-D-2Nal-Phe-Apc-NH₂ 811.00 810.5 96 50H-Apc-D-1Nal-D-1Nal-Phe-Lys-NH₂ 813.01 812.5 99 51H-Apc-D-Bal-D-1Nal-Phe-Apc-NH₂ 817.02 816.5 96 52H-Apc-D-Bal-D-2Nal-Phe-Apc-NH₂ 817.02 816.5 94 53H-Apc-D-Bal-D-1Nal-Phe-Lys-NH₂ 819.04 818.5 99 54H-Apc-D-Bal-D-2Nal-Phe-Lys-NH₂ 819.04 818.5 98 55H-Apc-D-1Nal-D-Trp-2Thi-NH₂ 679.84 679.2 98 56 H-Apc-D-Bal-D-Trp-Phe-NH₂679.84 679.3 99 57 H-Apc-D-1Nal-D-Trp-Taz-NH₂ 680.83 680.3 99 58H-Apc-D-Bal-D-Trp-2Thi-NH₂ 685.87 685.2 97 59 H-Apc-D-Bal-D-Trp-Taz-NH₂686.86 686.2 99 60 H-Apc-D-2Nal-D-Trp-2Thi-NH₂ 679.84 679.2 95 61H-Apc-D-2Nal-D-Trp-Taz-NH₂ 680.83 680.2 97 62H-Inp-D-1Nal-D-Trp-Taz-Apc-NH₂ 791.97 791.5 98 63H-Inp-D-Bal-D-Trp-Taz-Apc-NH₂ 798.00 797.4 99 64H-Apc-D-1Nal-D-Trp-Taz-Apc-NH₂ 806.99 806.5 99 65H-Apc-D-Bal-D-Trp-Taz-Apc-NH₂ 813.02 812.4 98 66H-Inp-D-2Nal-D-Trp(ψ)-Pim 610.77 611.4 99 67 H-Inp-D-1Nal-D-Trp(ψ)-Pim610.77 611.3 99 68 H-Inp-D-Bal-D-Trp(ψ)-Pim 616.79 617.3 99 69H-Aib-D-Ser(Bzl)-D-Trp(ψ)-Pim 564.69 565.3 99 70H-Inp-D-Trp-D-2Nal(ψ)-Pim 610.77 611.4 99Biological Assay

The activities of compounds of the invention at the GHS receptor can beand were determined using techniques such as those described in theexamples provided below. In different embodiments a ghrelin analog hasat least about 50%, at least about 60%, at least about 70%, at leastabout 80%, or at least about 90%, functional activity relative toghrelin as determined using one or more of the Functional Activityassays described below; and/or has an IC₅₀ greater than about 1,000 nM,greater than about 100 nM, or greater than about 50 nM, using theReceptor Binding assay described below. With respect to IC₅₀, greaterrefers to potency and thus indicates a lesser amount is needed toachieve binding inhibition.

Assays measuring the ability of a compound to bind a GHS receptor employa GHS receptor, a fragment of the receptor comprising a ghrelin bindingsite, a polypeptide comprising such a fragment, or a derivative of thepolypeptide. Preferably, the assay uses the GHS receptor or a fragmentthereof. A polypeptide comprising a GHS receptor fragment that bindsghrelin can also contain one or more polypeptide regions not found in aGHS receptor. A derivative of such a polypeptide comprises a GHSreceptor fragment that binds ghrelin along with one or more non-peptidecomponents.

The GHS receptor amino acid sequence involved in binding can be readilyidentified using labeled ghrelin or ghrelin structural or functionalanalogs and different receptor fragments. Different strategies can beemployed to select fragments to be tested to narrow down the bindingregion. Examples of such strategies include testing consecutivefragments about 15 amino adds in length starting at the N-terminus, andtesting longer length fragments. If longer length fragments are tested,a fragment binding ghrelin can be subdivided to further locate theghrelin binding region. Fragments used for binding studies can begenerated using recombinant nucleic acid techniques.

Binding assays can be performed using individual compounds orpreparations containing different numbers of compounds A preparationcontaining different numbers of compounds having the ability to bind tothe GHS receptor can be divided into smaller groups of compounds thatcan be tested to identify the compound(s) binding to the GHS receptor.In an embodiment of the present invention, a test preparation containingat least 10 compounds is used In a binding assay.

Binding assays can be performed using recombinantly produced GHSreceptor polypeptides present in different environments. Suchenvironments include, for example, cell extracts and purified cellextracts containing the GHS receptor polypeptide expressed from,recombinant nucleic acid or naturally occurring nucleic acid; and alsoinclude, for example, the use of a purified GHS receptor polypeptideproduced by recombinant means or from naturally occurring nucleic acidwhich is introduced into a different environment.

Screening for GHS Receptor Active Compounds

Screening for GHS receptor active compounds is facilitated using arecombinantly expressed receptor. Using a recombinantly expressed GHSreceptor offers several advantages such as the ability to express thereceptor in a defined cell system so that a response to a compound atthe GHS receptor can more readily be differentiated from responses atother receptors. For example, the GHS receptor can be expressed In acell line such as HEK 293, COS 7, and CHO not normally expressing thereceptor by an expression vector, wherein the same cell line without theexpression vector can act as a control.

Screening for compounds reducing GHS receptor activity is facilitatedthrough the use of a ghrelin functional analog in the assay. The use ofa ghrelin functional analog in a screening assay provides for GHSreceptor activity. The effect of test compounds on such activity can bemeasured to identify, for example, allosteric modulators andantagonists.

GHS receptor activity can be measured using different techniques such asdetecting a change in the intracellular conformation of the GHSreceptor, in the G-protein coupled activities, and/or in theintracellular messengers. Preferably, GHS receptor activity is measuredusing techniques such as those measuring intracellular Ca²⁺. Examples oftechniques well known in the art that can be employed to measure Ca²⁺include the use of dyes such as Fura-2 and the use ofCa²⁺-bioluminescent sensitive reporter proteins such as aequorin. Anexample of a cell line employing aequorin to measure G-protein activityis HEK293/aeq17. (Button et al., 1993. Cell Calcium 14, 663-671, andFeighner et al., 1999, Science 284, 2184-2188.)

Chimeric receptors containing a ghrelin binding region functionarycoupled to a different G-protein can also be used to measure GHSreceptor activity. A chimeric GHS receptor contains an N-terminalextracellular domain; a transmembrane domain made up of transmembraneregions, extracellular loop regions, and intracellular loop regions; andan intracellular carboxy terminus. Techniques for producing chimericreceptors and measuring G-protein coupled responses are provided in, forexample, International Application Number WO 97/05252, and U.S. Pat. No.5,264,565, both of which are hereby incorporated by reference herein.

Stimulation of GHS Receptor Activity

Structural and/or functional analogs of ghrelin can be used to stimulateGHS receptor activity. Such stimulation can be used, for example, tostudy the effect of GHS receptor modulation, to study the effect ofgrowth hormone secretion, to look for or study ghrelin antagonists, orto achieve a beneficial effect in a subject. Beneficial effects that canbe achieved include one or more of the following: treating a growthhormone deficient state, increasing muscle mass, increasing bonedensity, treating sexual dysfunction in males or females, facilitating aweight gain, facilitating maintenance of weight, facilitatingmaintenance of physical functioning, facilitating recovery of physicalfunction, and/or facilitating appetite increase.

Increasing weight or appetite can be useful for maintaining weight orproducing a weight or appetite gain in an underweight subject, or in apatient having a disease or undergoing treatment that affects weight orappetite. In addition, for example, farm animals such as pigs, cows andchickens can be treated to gain weight.

Underweight subjects include those having a body weight about 10% orless, 20% or less, or 30% or less, than the lower end of a “normal”weight range or Body Mass Index (“BMI”). BMI measures a subjectsheight/weight ratio and is determined by calculating weight in kilogramsdivided by the square of height in meters. BMI measures yourheight/weight ratio. It is determined by calculating weight in kilogramsdivided by the square of height in meters. The BMI “normal” range forhumans is generally considered to be 19-22. “Normal” weight ranges arewell known in the art and take into account factors such as a subjectage, height, and body type.

Biological Assays—Examples

1. Receptor Binding Assay

A. Preparation of CHO-K1 Cells Expressing the Human Recombinant GHSReceptor

The cDNA for human growth hormone secretagogue receptor (hGHS-R, orghrelin receptor) was cloned by Polymerase Chain Reaction (PCR) usinghuman brain RNA as a template (Clontech, Palo Alto, Calif.), genespecific primers flanking the full-length coding sequence of hGHS-R, (S:5′-ATGTGGAACGCGACGCC CAGCGAAGAG-3′(SEQ ID NO:1) and AS:5′-TCATGTATTAATAC TAGATTCTGTCCA-3′) (SEQ ID NO:2), and Advantage 2 PCRKit (Clontech). The PCR product was cloned into the pCR2.1 vector usingOriginal TA Cloning Kit (Invitrogen, Carlsbad, Calif.). The full lengthhuman GHS-R was subcloned into the mammalian expression vector pcDNA 3.1(Invitrogen). The plasmid was transfected into the Chinese hamster ovarycell line, CHO-K1 (American Type Culture Collection, Rockville, Md.), bycalcium phosphate method (Wigler, M et al., Cell 11, 223, 1977). Singlecell clones stably expressing the hGHS-R were obtained by selectingtransfected cells grown in cloning rings in RPMI 1640 media supplementedwith 10% fetal bovine serum and 1 mM sodium pyruvate containing 0.8mg/ml G418 (Gibco, Grand Island, N.Y.).

B. GHS-R Binding Assay:

Membranes for radioligand binding studies can be and were prepared byhomogenization of the foregoing CHO-K1 cells expressing the humanrecombinant GHS receptor in 20 ml of ice-cold 50 mM Tris-HCl with aBrinkman Polytron (Westbury, N.Y.) (setting 6, 15 sec). The homogenateswere washed twice by centrifugation (39,000 g/10 min), and the finalpellets were resuspended in 50 mM Tris-HCl, containing 2.5 mM MgCl₂, and0.1% BSA. For assay, aliquots (0.4 ml) were Incubated with 0.05 nM(¹²⁵I)ghrelin (˜2000 Ci/mmol, Perkin Elmer Life Sciences, Boston,Mass.), with and without 0.05 ml of unlabeled competing test compoundsof the invention. After a 60 min incubation (4° C.), the bound(¹²⁵I)ghrelin was separated from the free by rapid filtration throughGF/C filters (Brandel, Gaithersburg, Md.), which had been previouslysoaked in 0.5% polyethyleneimine/0.1% BSA. The filters were then washedthree times with 5-ml aliquots of ice-cold 50 mM Tris-HCl and 0.1%bovine serum albumin, and the bound radioactivity trapped on the filterswas counted by gamma spectrometry (Wallac L K B, Gaithersburg, Md.).Specific binding was defined as the total (¹²⁵I)ghrelin bound minus thatbound in the presence of 1000 nM ghrelin (Bachem, Torrence, Calif.).

2. GHS-R Functional Activity Assays

A. In Vitro GSH Receptor Mediated Intracellular iCa²⁺ Mobilization

The foregoing CHO-K1 cells expressing the human GSH receptor wereharvested by incubating in a 0.3% EDTA/phosphate buffered salinesolution (25° C.), and washed twice by centrifugation. The washed cellswere resuspended in Hank's-buffered saline solution (HBSS) for loadingof the fluorescent Ca²⁺ indicator Fura-2AM. Cell suspensions ofapproximately 10⁶ cells/ml were incubated with 2 μM Fura-2AM for 30 minat about 25° C. Unloaded Fura-2AM was removed by centrifugation twice inHBBS, and the final suspensions were transferred to a spectrofluorometer(Hitachi F-2000) equipped with a magnetic stirring mechanism and atemperature-regulated cuvette holder. After equilibration to 37° C., thecompounds of the invention were added for measurement of intracellularCa²⁺ mobilization. The excitation and emission wavelengths were 340 and510 nm, respectively.

B. In Vivo GH Release/Suppression

As is well known in the art, compounds may be tested for their abilityto stimulate or suppress release of growth hormone (GH) in vivo. (See,e.g., Deghenghi, R., et al., Life Sciences 54, 1321-1328 (1994);International Application No. WO 02/08250.) Thus for example in order toascertain a compound's ability to stimulate GH release in vivo thecompound may be injected subcutaneously in 10-day old rats at a dose of,e.g., 300 mg/kg. The circulating GH may be determined at, e.g., 15minutes after injection and compared to GH levels in rats injected witha solvent control.

Similarly, compounds may be tested for their ability to antagonizeghrelin-induced GH secretion in vivo. Thus a compound may be injectedsubcutaneously in 10-day old rats at a dose of, e.g., 300 mg/kg, alongwith ghrelin. Again the circulating GH may be determined at, e.g., 15minutes after injection and compared to GH levels in rats Injected withghrelin alone.

Administration

The compounds of the invention can be formulated and administered to asubject using the guidance provided herein along with techniques wellknown in the art. The preferred route of administration ensures that aneffective amount of compound reaches the target Guidelines forpharmaceutical administration in general are provided in, for example,Remington's Pharmaceutical Sciences 18^(th) Edition, Ed. Gennaro, MackPublishing, 1990, and Modern Pharmaceutics 2^(nd) Edition, Eds. Bankerand Rhodes, Marcel Dekker, Inc., 1990, both of which are herebyincorporated by reference herein.

The compounds of the invention can be prepared as acidic or basic salts.Pharmaceutically acceptable salts (in the form of water- or oil-solubleor dispersible products) include conventional non-toxic salts or thequaternary ammonium salts that are formed, e.g., from inorganic ororganic acids or bases. Examples of such salts include acid additionsalts such as acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartate, thiocyanate, tosylate, and undecanoate;and base salts such as ammonium salts, alkali metal salts such as sodiumand potassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such as arginineand lysine.

The compounds of the invention can be administered using differentroutes including oral, nasal, by injection, transdermal, andtransmucosally. Active ingredients to be administered orally as asuspension can be prepared according to techniques well known in the artof pharmaceutical formulation and may contain microcrystalline cellulosefor imparting bulk, alginic acid or sodium alginate as a suspendingagent, methylcellulose as a viscosity enhancer, and sweeteners/flavoringagents. As immediate release tablets, these compositions may containmicrocrystalline cellulose, dicalcium phosphate, starch, magnesiumstearate and lactose and/or other excipients, binders, extenders,disintegrants, diluents and lubricants.

Administered by nasal aerosol or inhalation formulations may beprepared, for example, as solutions in saline, employing benzyl alcoholor other suitable preservatives, absorption promoters to enhancebioavailability, employing fluorocarbons, and/or employing othersolubilizing or dispersing agents.

The compounds of the invention may also be administered in intravenous(both bolus and infusion), intraperitoneal, subcutaneous, topical withor without occlusion, or intramuscular form. When administered byinjection, the injectable solution or suspension may be formulated usingsuitable non-toxic, parenterally-acceptable diluents or solvents, suchas Ringer's solution or isotonic sodium chloride solution, or suitabledispersing or wetting and suspending agents, such as sterile, bland,fixed oils, including synthetic mono- or diglycerides, and fatty acids,including oleic acid.

Suitable dosing regimens are preferably determined taking into accountfactors well known in the art including type of subject being dosed;age, weight, sex and medical condition of the subject the route ofadministration; the renal and hepatic function of the subject; thedesired effect; and the particular compound employed.

Optimal precision in achieving concentrations of drug within the rangethat yields efficacy without toxicity requires a regimen based on thekinetics of the drug's availability to target sites. This involves aconsideration of the distribution, equilibrium, and elimination of adrug. The daily dose for a subject is expected to be between 0.01 and1,000 mg per subject per day.

The compounds of the invention can be provided in a kit Such a kittypically contains an active compound in dosage forms foradministration. A dosage form contains a sufficient amount of activecompound such that a desirable effect can be obtained when administeredto a subject during regular intervals, such as 1 to 6 times a day,during the course of 1 or more days. Preferably, a kit containsinstructions indicating the use of the dosage form to achieve adesirable affect and the amount of dosage form to be taken over aspecified time period.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims the inventionmay be practiced otherwise than as specifically described.

The patent and scientific literature referred to herein representsknowledge that is available to those with skill in the art. All patents,patent publications and other publications cited herein are herebyincorporated by reference in their entirety.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, that the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the claims.

1. A synthesized compound wherein said compound isH-Inp-D-Bal-D-Trp-Phe-Apc-NH₂, or a pharmaceutically acceptable saltthereof.
 2. A method of determining a compound's ability to bind to agrowth hormone secretagogue (GHS) receptor, said method comprising thestep of measuring the ability of a compound to affect binding of acompound according to claim 1 to said receptor, to a fragment of saidreceptor comprising a ghrelin binding site, to a polypeptide comprisingsaid fragment of said receptor, or to a derivative of said polypeptidecomprising said fragment of said receptor.